Beta-glucan-containing fat compositions and novel microorganism producing beta-glucan

ABSTRACT

A β-glucan-containing fat and oil composition contains β-glucan of microorganism origin or basidiomycete origin. The β-glucan-containing fat and oil composition has β-glucan uniformly dispersed in a food without worsening the texture, taste etc. of the food. The novel microorganism can efficiently produce β-glucan which has a high activity and favorable qualities as β-glucan of microorganism origin as described above from less expensive saccharides such as sucrose at a high production speed.

TECHNICAL FIELD

The present invention relates to a fat and oil composition containingβ-glucan originating in microorganisms or basidiomycetes. The fat andoil composition of the invention has β-glucan uniformly dispersed in fatand oil. Added to a food, etc., the composition provides the food withuniformly dispersed β-glucan having bioregulatory functions and alsowith enhanced taste, texture, and flavor.

The present invention also relates to a novel microorganism useful forobtaining β-glucan and a process of producing β-glucan using themicroorganism.

Background Art

Beta-glucans are material attracting attention for the utility becauseof their excellent bioregulatory functions that have recently beenanalyzed, such as lipid metabolism improving action, intestinalregulatory action, blood sugar controlling action, antitumor effect, andimmune enhancing action. Application of such material to a broad rangeof processed foods will bring extreme benefits, not only contributing toenhancement of functionality of processed foods (addition of value) butmatching the expectation of contribution to public health maintenance.Beta-glucans occur in a variety of organisms, including microorganisms,basidiomycetes, and plants, chiefly constituting the skeleton of theorganisms. Beta-glucans, for the most part, serve to make up cell walls.Beta-glucans are composed mainly of glucose polymers having at least twokinds of β-1-2,1-3, 1-4, and 1-6-D-glucopyranose bonds.

JP-T-2001-501996 studies β-glucans derived from grains and gramineousplants. Some β-glucans from grains and gramineous plants containpolyphenols, which can cause a coloration problem. Moreover, theβ-glucans from these origins are expensive due to low original β-glucancontents, which puts a limit on applicability to foods.

Among microorganisms and basidiomycetes there are strains which secretethe same β-glucan out of fungi as their cell wall component under somecultivation conditions. The cell wall of microorganisms andbasidiomycetes contain a large quantity of β-glucan.

Beta-glucan of microorganism or basidiomycete origin, including β-glucansecreted out of fungi by microorganisms or basidiomycetes, β-glucanharvested from microorganisms or basidiomycetes by isolation,extraction, purification or like means, cell wall components ofmicroorganisms or basidiomycetes, and fungi per se, can or could beadded to processed foods, for example, as follows. (1) Culture fungicollected from the culture of microorganisms or basidiomycetes are addeddirectly to raw materials of processed foods. (2) Cell wall componentsof microorganisms or basidiomycetes separated from the culture andpurified are added to raw materials of processed foods. (3) Beta-glucanextracted from fungi or separated cell wall components is added to rawmaterials of processed foods. (4) The supernatant liquid of a culture ofmicroorganisms or basidiomycetes or β-glucan separated and purified fromthe supernatant liquid is added to raw materials of processed foods.

In view of the fact that many of β-glucans are polymers having amolecular weight of 10,000 or more, and some of them are sparinglysoluble in water, it is very difficult to uniformly mix β-glucan withmaterials of processed foods by the method (1) or (2). Processed foodshaving β-glucan added thereto by the method (1) or (2) suffer fromimpairment of texture or reduction of commercial value, such as unevenbaking.

The methods (3) and (4) are advantageous in that β-glucan can beincorporated into processed foods relatively uniformly and that theβ-glucan content in processed foods can be adjusted freely. However, theextracted and purified β-glucan has problems arising from its high waterabsorptivity. If such β-glucan is added as such to, for example, a doughmix containing wheat flour as a main ingredient, and the mix is kneadedtogether with water, the β-glucan forms lumps to make non-uniform dough,which results in processed food products with reduced taste and textureand reduced quality. When β-glucan previously dissolved in water isadded to a dough mix (mostly in powder form), the resultingβ-glucan-containing foods can have β-glucan dispersed therein relativelyuniformly. In this case, however, dissolving β-glucan in water needsmuch time, the aqueous solution takes on viscosity, and it is not easyto obtain a uniform aqueous solution. Accordingly, to dissolve in wateris an impractical operation that impairs the workability on site.

It has therefore been awaited to establish a convenient process forproducing processed foods in which β-glucan of microorganism orbasidiomycete origin is uniformly dispersed and to develop such aβ-glucan material.

Beta-glucans activating the immune system include plant cell wallcomponents (see JP-B-62-6692 and JP-A-2001-323001), those present in thehymenia and the mycelia of basidiomycetes (mushrooms) (see K Sasaki etal., Carbohydrate Res., vol. 47, 99-104 (1976) and JP-A-5-345725), cellwall components of microbial fungi, and those secreted and produced outof fungi.

It is generally well known that the cell wall components of anymicroorganisms contains β-glucans and exhibit immune enhancement. Amongthem yeast fungi (see JP-A-54-138115 and JP-A-9-103266), lactic acidbacterial fungi (see JP-A-3-22970 and JP-A-10-167972), fungus ofAureobasidium (see JP-B-6-92441), etc. are known to be of high safetyand high utility value as foods.

Microorganisms that are known to secrete and produce β-glucan out offungi exhibiting immune system enhancement include the genusMacrophomopsis, the genus Alcaligenes producing curdlan (seeSyokumotusen-i no kagaku, Asakura Shoten, 1977, 108), and Aureobasidiumpullulans (see Agaric. Biol. Chem., 47(6), 1983, 1167-1172 andJP-A-6-340701).

The β-glucans present in the hymenia and the mycelia of basidiomycetes(mushrooms) have high immune enhancing activity, and some of them,exemplified by lentinan extracted from the hymenia of Lentinus edodes,have been made use of as medicines. In general, however, production ofβ-glucan by the hymenia and the mycelia of basidiomycetes (mushrooms)greatly varies depending on the growth or cultivation conditions, and itis necessary to separate the β-glucan by extraction. As a result,complicated and wide-ranging molecular species of β-glucan are found inthe resulting extract, including both high-molecular-weight ones andlow-molecular-weight ones, which means unstable quality.

Thus, stable production of β-glucan with constant high activity is thestanding problem relating to basidiomycetes (mushrooms). On the otherhand, the cell walls of microorganisms contain a large amount ofβ-glucan and are of interest as a source of β-glucan. Nevertheless,because the cell walls of microorganisms contain other components thanβ-glucan and are insoluble in water, they need an extraction operationsimilarly to basidiomycetes in order to recover highly effective,water-soluble β-glucan. It has therefore been an issue to establish aprocess for stably extracting β-glucan of constant quality.

In contrast, the fermentation method of producing β-glucan usingmicroorganisms secreting and producing water-soluble β-glucan out offungi with high immune enhancing activity is an extremely effectivetechnology, making it feasible to obtain uniform, water-soluble, andhigh-activity β-glucan. Along this line, processes of producing β-glucanusing a microorganism belonging to the genus Aureobasidium which isknown to secrete and produce high-activity β-glucan out of fungi havebeen proposed.

Microorganisms belonging to the genus Aureobasidium are known, however,to secrete and produce pullulan, α-glucan, out of fungi when culturedusing carbon sources commonly used in microbial cultivation, such assucrose (see JP-B-51-36360 and JP-B-51-42199). This has made productionof high purity β-glucan difficult (see JP-A-06-340701). Moreover,microorganisms of the genus Aureobasidium are also called “black yeast”.Black yeast produces melanin pigment, with which the fungi or theculture solution are pigmented in black, and so is the resultingβ-glucan. Therefore, the produced β-glucan has seriously ruined productquality. To address this problem, it has been proposed to generate amutant that does not involve pigmentation by mutagenic treatment and toproduce polysaccharides including pullulan by using the mutant(JP-B-4-18835). However, no strain is known that is capable of secretingand producing β-glucan of high purity with good efficiency out of fungi(but incapable of secreting and producing pullulans out of fungi)completely without involving melanin production.

Culturing techniques for the production of β-glucan in whichby-production of pullulan, regarded as impurity, during culturing issuppressed have also been studied. JP-A-6-340701 and JP-A-07-51080propose a process in which pullulan production is controlled by pHadjustment or use of specific sugar as a carbon source thereby toprovide β-glucan at high purity. The problem of this process for theproduction of β-glucan is that the operation is cumbersome becausespecial culturing conditions should be set or that the medium is costlybecause a special carbon source should be used.

Accordingly, for the production of β-glucan by the use of microorganismsof the genus Aureobasidium, a strain is needed that produces no, or alimited amount of, impurity such as pullulan even when cultured usinginexpensive saccharides commonly employed in microbial cultivation as acarbon source and that involves substantially no or limited productionof pigment melanin during production of β-glucan, thereby secreting andproducing high-activity and high-quality β-glucan with good efficiencyout of fungi.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a β-glucan materialthat provides a food with uniformly dispersed β-glucan havingbioregulatory functions without impairing the taste and texture of thefood. Another object of the invention is to provide a novelmicroorganism that is useful for producing high-activity andhigh-quality β-glucan from inexpensive saccharides such as sucrose withgood efficiency and at high production rates, a process of producingβ-glucan using the microorganism, and β-glucan secreted and produced outof fungi by the microorganism.

As a result of extensive investigations, the present inventors havenoted use of β-glucan of microorganism or basidiomycete origin and foundthat dispersing the β-glucan in fats and oils or fat and oilcompositions gives β-glucan materials that accomplish the above objects.

Furthermore, for the purpose of accomplishing the above objects, thepresent inventors have sought for a microorganism capable of secretingand producing high-purity β-glucan out of fungi efficiently. As a resultthey have found a novel microorganism secreting and producinghigh-quality β-glucan out of fungi at high efficiency. As a result offurther researches, they have reached the finding that a strainbelonging to the genus Aureobasidium and resistant to the antibioticcycloheximide is capable of secreting and producing β-glucan out offungi with high purity at high efficiency.

The present invention provides a β-glucan-containing fat and oilcomposition characterized by containing β-glucan of microorganism orbasidiomycete origin.

The present invention also provides a microorganism of which the 18SrRNA gene contains the sequence of 1732 bases shown in Sequence Listing,SEQ ID No. 1 or a base sequence molecular-phylogenetically equivalentthereto based on the 18S rRNA gene base sequence and which hasresistance to the antibiotic cycloheximide and is capable of secretingand producing β-glucan out of fungi.

The present invention also provides a microorganism of which theITS-5.8S rRNA gene contains the sequence of 563 bases shown in SequenceListing, SEQ ID No. 2 or a base sequence molecular-phylogeneticallyequivalent thereto based on the ITS-5.8S rRNA gene base sequence andwhich is capable of secreting and producing β-glucan out of fungi. It ispreferred for this microorganism to have resistance to the antibioticcycloheximide.

The present invention also provides the above-described microorganismswhich are capable of secreting and producing β-glucan having at least aβ-1,3-D-glucopyranose bond in the structure out of fungi thereof.

The present invention also provides the above-described microorganismswhich belong to the genus Aureobasidium.

The present invention also provides the above-described microorganismwhich is Aureobasidium pullulans ADK-34 (FERM BP-8391).

The present invention also provides a process of producing β-glucancomprising culturing any of the above-described microorganisms(preferably in a culture medium containing a saccharide as a carbonsource), secreting and producing β-glucan out of fungi.

The present invention also provides a process of producing β-glucancomprising culturing a microorganism of which the ITS-5.8S rRNA geneexhibits sequence homology of at least 98% with the base sequence shownin Sequence Listing, SEQ ID No. 2 (preferably in a culture mediumcontaining a saccharide as a carbon source), secreting and producingβ-glucan out of fungi.

The present invention also provides β-glucan which is secreted andproduced out of fungi by culturing Aureobasidium pullulans ADK-34 (FERMBP-8391) and has at least a β-1,3-D-glucopyranose bond in the structurethereof and a fat and oil composition containing the β-glucan.

The present invention also provides a food containing theabove-described β-glucan-containing fat and oil composition according tothe present invention and a drug containing the above-describedβ-glucan-containing fat and oil composition according to the presentinvention and having preventive activities on habitual diseases (orlife-style related diseases).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will now be illustrated in detail.

The β-glucan that can be used in the fat and oil composition of thepresent invention is one originating in a microorganism or abasidiomycete, i.e., one obtained from a microorganism or abasidiomycete.

The β-glucan of microorganism origin useful in the present invention isdescribed first.

Since microbial cells per se contain a large amount of β-glucan in theirwalls, the “β-glucan of microorganism origin” may be culture cells asobtained by inoculating microorganisms into the respective growth mediafollowed by fungous proliferation or a culture cell wall residueobtained by disrupting the culture cells and removing the cell contents.The “β-glucan of microorganism origin” can also be one extracted fromthe culture cells or the culture cell wall residue, either as such or aspurified. It is also possible to use β-glucan secreted and produced outof fungi by microorganism culturing. In this case, the culture solutionafter cultivation can be used as such, or β-glucan may be isolatedtherefrom and purified.

Where the culture cells as obtained by inoculating microorganisms intothe respective growth media followed by fungous proliferation are usedas they are, the cell contents can reduce the taste or texture or thephysical properties of a processed food to which the fat and oilcomposition is added. It is therefore desirable to use the culture cellwall residue obtained by disrupting the culture cells and removing thecell contents. It is more desirable to use the β-glucan extracted fromthe culture cells or the culture cell wall residue either as such or aspurified. It is the most desirable to use the secreted and producedβ-glucan out of fungi either together with the culture solution or afterisolated and purified.

Microorganisms suitable for obtaining β-glucan are those that havehitherto been used in food industries and proved highly safe. Suchmicroorganisms include yeasts, lactic acid bacteria, natto bacteria,acetic acid bacteria, koji bacteria, algae, such as Chlorella andSpirulina, and microorganisms belonging to the genus Aureobasidium.These microorganisms can be those isolated from the environment (such asa food, soil or a room). Stock strains or isolated strains or mutantsobtained from stock strains or isolated strains by ordinary mutationoperations can be used. Useful mutation operations include UVirradiation and treatments with chemicals such as nitrosoguanidine,ethidium bromide, methane ethyl sulfonate, and sodium nitrite.

The yeasts include those classified into the genus Saccharomyces thatare used in making alcohols (such as beer, carbonated alcohols, shochu(Japanese distilled liquor), sake, wine, and whisky) or bread;compressed yeast; those used in making soy sauce; and those of the genusCandida used in the production of single-cell protein.

The lactic acid bacteria that are usually used include bacilli, such asthose of genera Lactobacillus and Bifidobacterium, and cocci, such asthose of genera Leuconostock, Pediococcus, Streptococcus, andLactococcus. In addition, lactic acid bacteria of the generaEnterococcus, Vagococcus, Carnobacterium, Aerococcus, andTetragenococcus are also useful. More specifically, one or more of thefollowing traditionally utilized lactic acid bacteria species can beused: Lactobacillus bulgaricus, L. helveticus, L. acidophilus, L.lactis, L. casei, L. brevis, L. plantarum, L. sake, Streptococcusthermophilus, S. lactis, S. cremoris, Bifidobacterium longum, B.bifidum, B. breve, B. infantis, Leuconostoc cremoris, Ln. mesenteroides,Ln. ocnos, Pediococcus acidilactici, P. cerevisiae, and P. pentosaceus.These species can be used either individually or as a combination of twoor more thereof. A lactic acid bacterium of the genus Bifidobacteriumand another lactic acid bacteria may be cultured separately, and thecultures may be mixed.

The microorganisms belonging to the genus Aureobasidium are not limitedas long as they are capable of producing glucose polymers having aβ-bond out of fungi when cultured. Strains of Aureobasidium pullulans,e.g., IFO 4467, IFO 4466, IFO 6353, IFO 7757, ATCC 9348, ATCC 3092, ATCC42023, and ATCC 433023, are mentioned as examples. The microorganismsisolated from the environment (e.g., foods, soil, rooms) can be used.Stock strains or isolated strains or mutants obtained from stock strainsor isolated strains by ordinary mutation operations can also be used.Useful mutation operations include UV irradiation and treatments withchemicals such as nitrosoguanidine, ethidium bromide, methane ethylsulfonate, and sodium nitrite.

Additional microorganisms that can be used include natto bacteria of thegenus Bacillus, acetic acid bacteria of the genus Acetobactor, kojibacteria of the genus Aspergillus, bacteria of the genus Penicillium,algae, e.g., Chlorella and Spirulina, dry chlorella powder, strains ofthe genus Aureobasidium known capable of secreting and producingpullulan out of fungi, and strains of the genera Xanthomonas, Aeromonas,Azotobactor, Alcaligenes, Erwinia, Enterobactor, Sclerotium,Pseudomonas, Agrobacterium, and Macrophomopsis known capable ofproducing thickening polysaccharides useful as food additives.

The microorganisms that are suited for use in the present inventioninclude the novel ones according to the invention, i.e., a microorganismof which the 18S rRNA gene contains the sequence of 1732 bases shown inSequence Listing, SEQ ID No. 1 or a base sequencemolecular-phylogenetically equivalent thereto based on the 18S rRNA genebase sequence and which is resistant to the antibiotic cycloheximide andcapable of secreting and producing β-glucan out of fungi and amicroorganism of which the ITS-5.8S rRNA gene contains the sequence of563 bases shown in Sequence Listing, SEQ ID No. 2 or a base sequencemolecular-phylogenetically equivalent thereto based on the ITS-5.8S rRNAgene base sequence and which is capable of secreting and producingβ-glucan out of fungi. The novel microorganisms of the invention will bedescribed later in greater detail.

The β-glucan of basidiomycete origin that can be used in the presentinvention is then described hereunder.

Since basidiomycetes contain a large amount of β-glucan in the hymeniumand the sclerotium, a compact mass of hyphae, either of finely groundhymenium or sclerotium and an extract from the ground hymenium orsclerotium, either as such or as purified, can be used as β-glucan ofbasidiomycete origin. Culture cells obtained by germinating spores ofbasidiomycetes, inoculating the mycelia into the respective culturemedia, followed by fungous proliferation can also be used as such. Aculture cell wall residue prepared by disrupting the culture cells andremoving the cell contents can also be used. Beta-glucan extracted fromthe culture cells or the culture cell wall residue can also be used asβ-glucan of basidiomycete origin, either as such or after purification.It is also possible to use β-glucan secreted and produced out of fungiby culturing basidiomycetes. In this case, the culture solution aftercultivation can be used as such or after isolation and purification ofthe β-glucan.

Where the finely ground hymenium or sclerotium or the β-glucan extractedtherefrom or the culture cells as obtained by inoculating spores ormycelium into the respective growth media followed by fungousproliferation are used as they are, the cell contents can reduce thetaste or texture or the physical properties of a processed food to whichthe fat and oil composition is added. It is therefore desirable to usethe culture cell wall residue obtained by disrupting the culture cellsand removing the cell contents. It is more desirable to use the β-glucanextracted from the culture cells or the culture cell wall residue eitheras such or as purified. It is the most desirable to use the secreted andproduced β-glucan out of fungi either together with the culture solutionor after separated from the culture solution and purified.

While the most preferred basidiomycetes for use in the invention arecultivated varieties, β-glucan from basidiomycetes that are not incommercial production are useful as well. Useful species includeAgaricus blazei, Morchella esculenta (common morel), Suillus bovinus,Climacodon septentrionalis, Flammulina velutipes, Fistulina hepatica,Auricularia auricula, Dictyophora indusiata, Naematoloma sublateritium,Stropharia rugosoannulata, Coprinus comatus, Hericium ramosum, Lentinusedodes, Rhizopogon rubescens, Tremella fuciformis, Lyophyllum ulmayium,Pleurocybella porrigens, Pleurotus cornucopiae, Polyporus umbellatus,Pleurotus dryinus, Cordyceps, Pholiota nameko, Armillariella mellea,Armillariella tabescens, Tricholoma giganteum, Gloeostereum incarnatum,Pseudohydnum gelatinosum, Pholiota adiposa, Pholiota aurivella,Lactarius hatsudake, Pleurotus ostreatus, Wolfiporia cocos, Volvariellavolvacea, Hypsizigus marmoreus, Myceleptodonoides aitchisonii,Lyophyllum shimeji, Grifola frondosa, Laetiporus sulphureus, Lentinuslepideus, Agaricus bisporus, Tricholoma matsutake, Ganoderma lucidum,Panellus serotinus, Lepista nuda, Boletus edulis, Hericium erinaceum,and Agrocybe cylindracea.

A culture cell wall residue as β-glucan of the above-recitedmicroorganisms and basidiomycetes can be obtained by, for example, amethod comprising adding an adequate amount of a solvent to culturedmicroorganisms, cultured mycelia, cultured sclerotia or culturedhymenia, destroying part of the cell walls by autodigestion or byaddition of a hydrolase to allow the contents to flow out, andcollecting the residue as β-glucan or a method comprising applying aphysical force by a French press, an ultrasonic processor, etc. to thecells to destroy part of the cells, removing the cell contents, andcollecting the residue as β-glucan.

Beta-glucan can be extracted by any method. Extraction is effected byadding an extracting solvent to the extraction material, themicroorganism or basidiomycete. Usable extracting solvents includewater, salt solutions, aqueous acid solutions, aqueous alkali solutions,organic solvents, and mixtures of two or more thereof. A combined use ofa cell wall degrading enzyme will increase the extraction efficiency.The extract may be used in any form and at any purity. That is, theextract separated by solid-liquid separation of the extraction systemmay be used as such, or the extract may be concentrated in a usualmanner to liquid or solid, or the extract may be purified in a knownmanner into liquid or solid with increased purity. It does not matter ifthe extract contains any extracted component other than β-glucan. In thepresent invention all these product forms are called β-glucan extractedfrom microorganisms or basidiomycetes.

The method of extracting β-glucan from microorganisms or basidiomyceteswill be described further. The β-glucan used in the invention is awater-soluble polymer dissolving in a solvent such as water. Forexample, a commercially available mushrooms, i.e., a basidiomycete isdried and ground to powder, and the powder is extracted with 2 to 100times the amount of water, warm water, hot water, a salt solution, anaqueous acid or alkali solution, an organic solvent, etc. at anarbitrary temperature for an arbitrary period of time. The extractionsystem is separated into liquid and solid to recover the extractedβ-glucan. Beta-glucan extracted with water, warm water or hot water ispreferred. Beta-glucan extracted with water at temperature of 4° to 80°C. is still preferred. An extraction accelerator, such as an enzymesolution, may be added to the system.

The β-glucan used in the invention is preferably one having at least twokinds of a β-1-2-D-glucopyranose bond, a β-1-3-D-glucopyranose bond, aβ-1-4-D-glucopyranose bond, and a β-1-6-D-glucopyranose bond.Beta-glucan having a β-1,3-D-glucopyranose bond and aβ-1,4-D-glucopyranose bond, one having a β-1,3-D-glucopyranose bond anda β-1,6-D-glucopyranose bond, and one having a β-1-3-D-glucopyranosebond, a β-1-4-D-glucopyranose bond, and a β-1-6-D-glucopyranose bond areparticularly preferred. It should be noted that so-called curdlancomposed of β-1,3-D-glucopyranose bonds is not included under theβ-glucan of the invention.

The β-glucan used in the invention is a high-molecular-weight compound.While β-glucan having any weight average molecular weight is usable, onehaving a molecular weight of less than 3,000,000, preferably less than500,000, still preferably less than 100,000 is suitable because thecompatibility with fats and oils increases as the molecular weightdecreases. The molecular weight of extracted β-glucan may be reduced ina conventional manner to have improved compatibility with fats and oils,or low-molecular β-glucan may be directly obtained by extraction.

The fats and oils or fat and oil compositions in which the β-glucan ofmicroorganism or basidiomycete origin or originating in the novelmicroorganisms according to the invention is dispersed are notparticularly limited as long as they are edible. Examples are rice oil,rapeseed oil, soybean oil, cotton seed oil, palm oil, palm kernel oil,coconut oil, olive oil, fish oil, beef tallow, lard, cacao butter;processed oils derived from these fats and oils according to necessity,such as hydrogenated oils, slightly hydrogenated oils, hydrogenationisomerized oils, interesterified oils, and fractionated oils; fats andoils processed by two or more of the recited processes; and mixtures oftwo or more of these fats and oils. Additionally, disperse systems usingthese edible fats and oils as a dispersing medium or a dispersoid, suchas emulsions (including W/O emulsions, O/W emulsions, double emulsions,i.e., O/W/O emulsions and W/O/W emulsions, and more complex multipleemulsions) and suspensions, are also useful (such disperse systems willhereinafter be included under the category “fats and oils”).

The β-glucan-containing fat and oil composition of the present inventionis obtained by adding the β-glucan to the above-recited fat and oil,followed by mixing.

When added to fats and oils, the form of the β-glucan is notparticularly limited and can be added as such or as dissolved in wateror any other water-soluble solvents.

Where the fat and oil is an emulsion, the β-glucan may be dispersed in apreviously prepared fat and oil emulsion or be dispersed at the time ofemulsification. While it is possible to add the β-glucan to either theoily phase or the aqueous phase, it is preferred that the β-glucan befirst dispersed in the oily phase and then mixed with the aqueous phase.By so doing, the β-glucan will exhibit satisfactory compatibility withfat and oil to give a homogeneous β-glucan-containing fat and oilcomposition in a shorter time.

Where the fat and oil is a water-in-oil emulsion, a plasticizedwater-in-oil emulsion, etc., addition of the β-glucan may be followed byemulsification, or be simultaneous with emulsification, or be precededby emulsification as stated above, or be preceded by plasticization. Inusing solid fat, it may be softened or liquefied by an appropriatemethod according to necessity before mixing the β-glucan. In order tohighly uniformly disperse the β-glucan, it is desirable that 100 partsby weight of powdered β-glucan and 10 to 50 parts by weight of fat andoil are mixed, and the mixture is then subjected to rolling or acombination of rolling and conching. Other raw materials, an additionalamount of oil or fat, and the like may be added in this stage to adjustthe β-glucan content in the final β-glucan-containing fat and oilcomposition.

While the manner of the mixing following the addition of the β-glucan tothe fat and oil is not particularly restricted, it is advisable tomaintain the mixture of the β-glucan and the fat and oil at 50° C. orhigher for a given period of time, preferably 5 minutes to 6 hours,still preferably 10 minutes to 2 hours, thereby to obtain aβ-glucan-containing fat and oil composition in which the β-glucan isuniformly dispersed with sufficient compatibility with the fat and oil.Foods prepared using the thus prepared β-glucan-containing fat and oilcomposition have the β-glucan dispersed therein more uniformly thanthose prepared by directly adding β-glucan to foodstuffs. As a result,there are observed remarkable effects such that the taste or texture isnot impaired, suppression of flavor due to use of an emulsifier isunexpectedly reduced, and the flavor of foodstuffs is brought out more.

The means for mixing β-glucan into fats and oils include various typesof machines for mixing, kneading or stirring. Examples are propelleragitators, oscillatory mixers, orifice mixers, paddle agitators,agitation emulsifiers (homomixer), cutter mixers, cokneaders, conches,silent cutters, jet mixers, vacuum agitators, screw mixers, staticmixers, cutting mixers,

ultrasonic emulsifiers, kneaders, rolls, Hydrossure, pipeline mixers,universal mixers, pin machines, homogenizers (high-pressurehomogenizers), ball cutters, and ribbon mixers. It is preferred to usean agitation emulsifier (homomixer) and/or a homogenizer (high-pressurehomogenizer) at a product temperature of 40° to 80° C.

After mixing the β-glucan and the fat and oil by agitation, theresulting β-glucan-containing fat and oil composition may be stored asobtained, or emulsified, or rapidly cooled for plasticization. Forplasticization, a votator, a combinator, a perfector, a complector,Onreitor, etc. can be used. Use of a pin machine at a producttemperature of 10° C. or lower is preferred. It is also possible thatthe fat and oil is previously emulsified and processed in a rapidcooling-plasticizing apparatus, such as a votator, a combinator, aperfector, a complector or Onreitor, and the β-glucan is then added,followed by mixing by any of the above-described methods to prepare aglucan-containing fat and oil composition.

The β-glucan content in the fat and oil composition of the presentinvention is preferably 0.01 to 500 parts by weight, still preferably0.1 to 150 parts by weight, particularly preferably 1 to 100 parts byweight, per 100 parts by weight of the total of the components otherthan the β-glucan. Where the β-glucan content is less than 0.01 parts byweight, a final product tends to fail to exhibit the functional effectsof β-glucan. If it exceeds 500 parts by weight, the mixture tends tobecome powdery or lumpy irrespective of the kinds of other ingredients,failing to provide an edible fat and oil composition having a β-glucanmixed and dispersed therein uniformly. Even after the mixture isprocessed into a final product, it is very likely that the lumps wouldremain only to cause non-uniform distribution of the β-glucan.

Where an extract from a microorganism or a basidiomycete is used as suchwithout being purified or merely after being powdered or solidified, anacceptable purity of the β-glucan in the extract ranges 1 to 100%. Apreferred purity is from 10 to 100%, particularly 20 to 100%. The higherthe better.

It is possible to add, to the β-glucan-containing fat and oilcomposition of the present invention, food additives such asemulsifiers, gelling agents, thickeners, and stabilizers so as to ensurepreventing the β-glucan from getting distributed non-uniformly due toagglomeration into lumps in the composition. The food additives are notparticularly limited as far as they are edible. Examples of theemulsifiers are lecithin, fatty acid monoglycerides, sorbitan fatty acidesters, propylene glycol fatty acid esters, and sugar esters. Examplesof the thickeners and the stabilizers are pullulan, psyllium, gumarabic, gellan gum, glucomannan, guar gum, xanthan gum, tamarind gum,carrageenan, alginic acid salts, farceran, locust bean gum, pectin,curdlan, and low-molecular compounds derived from these substances;starch, processed starch, gelatinized starch, crystalline cellulose,gelatin, dextrin, agar, and dextran. Additional useful food additivesinclude saccharides, such as glucose, fructose, sucrose, maltose,enzyme-saccharified sugar (thick malt syrup), lactose, reducing starchsaccharification products, isomerized liquid sugar, sucrose-coupled maltsyrup, oligosaccharides, reducing sugar polydextrose, sorbitol, reducedlactose, trehalose, xylose, xylitol, maltitol, erythritol, mannitol,fructo-oligosaccharides, soybean oligosaccharides,galacto-oligosaccharides, lactosucrose-oligosaccharides, raffinose,lactulose, palatinose-oligosaccharides, stevia, and Aspartame;stabilizers, such as phosphoric acid salts (e.g., hexametaphosphates,secondary phosphates and primary phosphates), and alkali metal (e.g.,potassium or sodium) salts of citric acid; proteins, such as wheyproteins, (e.g., α-lactalbumin, β-lactoglobulin, and serum albumin),casein and other milk proteins, low-density lipoprotein, high-densitylipoprotein, egg proteins (e.g., phosvitin, livetin,phosphoglycoprotein, ovalbumin, conalbumin, and ovomucoid), wheatproteins (e.g., gliadin, glutenin, prolamine, and glutelin), and othervegetable and animal proteins; inorganic salts, such as sodium chloride,rock salt, sea salt, and potassium chloride; souring agents, such asacetic acid, lactic acid, and gluconic acid; colorants, such asβ-carotin, caramel, and Monascus color; antioxidants, such as tocopheroland tea extract; eggs, such as whole eggs, egg yolk, egg white, andenzyme-processed eggs; cereals, such as bread flour, all-purpose flour,and cake flour; beans, such as soybean powder; water, flavors, dairyproducts, seasonings, pH adjustors, enzymes, food preservatives, shelflife extenders, fruits, fruit juices, coffee, nut pastes, spices, cacaomass, and cocoa powder. Two or more of these additives can be used incombination. The amounts of the additives to be added are notparticularly limited. They can be added in general amounts, for example,0.01 to 15% by weight based on the composition.

The foods according to the present invention will now be describedhereunder. The foods of the invention contain the aforementionedβ-glucan-containing fat and oil composition as a part or the whole ofthe fat and oil ingredients conventionally employed therein. Included insuch foods are not only fat and oil foods exemplified by margarine andshortening but any kinds containing fats or oils, such as bakeryproducts, confectionery products, processed rice products, processedwheat products, processed maize products, processed soybean products,health foods, and medicinal foods. The β-glucan-containing fat and oilcomposition of the present invention substitutes for a part of, or thewhole of, fats and oils in these foods to provide foods that can beserved in a conventional manner irrespective of whether the foods areliquid (e.g., salad oil, frying oil, and whipping cream), sol (e.g.,liquid shortening), paste or emulsion (e.g., foamable emulsified fats,dressings, fat spreads, custard cream, and dipping cream) or solid(e.g., shortening, margarine, candies, chocolates, and roux type currysauce mixes).

The bakery products of the present invention are described below. Thebakery products contain the aforementioned β-glucan-containing fat andoil composition. The bakery products are produced by baking doughprepared by substituting a part or the whole of fats and oilsconventionally employed in making bakery products by theβ-glucan-containing fat and oil composition. The bakery products includebreads, pies, kasutera (Japanese style sponge cake), sponge cakes,butter cakes, puff pastries, waffles, and fermented confections. Themethod of preparing dough is not particularly limited, except that apart of or the whole of the fat and oil which have been conventionallyused in dough preparation is substituted with the β-glucan-containingfat and oil composition of the present invention. In making bread as anexample of the bakery products, bread dough is prepared from common rawmaterials of bread, such as wheat flour, water, yeast, sugar, and ediblesalt, and the β-glucan-containing fat and oil composition of theinvention by a known dough preparation method. For instance, aftermixing the other materials, the β-glucan-containing fat and oilcomposition is folded into the mix. The resulting dough is thenfermented, shaped, and baked in a usual manner. Similarly, theβ-glucan-containing fat and oil composition of the invention can be usedas a substitute for a part of or the whole of fat and oil for folding(roll-in fat and oil) or fat and oil for dough and batter in makingfolded pies; a part of or the whole of pieces of fat and oil in the formof chips, straws, etc. in making pie crusts; or a part of or the wholeof foamable emulsified fat and oil or liquid oil for cakes in makingsponge cakes.

Where the production of bakery products involves a baking step, if amicroorganism or a basidiomycete is added as such as a β-glucan sourceor even when β-glucan is directly added to dough or a powder mix, whichis then kneaded into dough, lumps are easily formed in the dough. Doughcontaining such lumps only provides a bakery product with a rough orgrainy texture or a strange texture due to non-uniform moisturedistribution or non-uniform firmness. To the contrary, use of theβ-glucan-containing fat and oil composition of the invention providesdough having the β-glucan uniformly dispersed therein with very fewlumps, which finally gives a baked product with a good texture, not onlyfree from a strange texture but with greatly improved softness.

The confectionery products are then described. The confectioneryproducts contain the aforementioned β-glucan-containing fat and oilcomposition of the invention. The confectionery products are produced byprocessing dough prepared by substituting a part or the whole of fatsand oils conventionally employed in making confectionery products withthe β-glucan-containing fat and oil composition. The confectioneryproducts include deep-fried products such as snacks and doughnuts andsteamed products such as steamed cakes and bean-jam buns. Another typeof the confectionery products includes candies, gums, chocolates, andtablets prepared by mixing the β-glucan-containing oil and fatcomposition with sugar, flavors, and the like and, if necessary,solidifying and molding the mixture. Cold desserts, such as sherbet, arealso included under confectionery products.

In making confectionery products, where the weight is put on not onlyflavor but taste, particularly sweetness, it is more important toeliminate lumps. Even a very small lump would cause a strange feelingand ruin the commercial value. Since the β-glucan-containing fat and oilcomposition according to the present invention previously containsβ-glucan in a uniformly dispersed state, even when it is added to andmixed with a premixed material, there is provided a final confectioneryproduct containing the β-glucan in a uniformly dispersed state with nolumps and having a good flavor with no strange taste.

The β-glucan-containing fat and oil composition according to the presentinvention can be added to foods or drugs containing a food ingredienthaving a prophylactic action for habitual diseases to enhance the actionof the foods or drugs. Such foods or drugs include those containingunsaturated higher fatty acids regulating a blood lipid concentration(e.g., EPA and DHA), plant sterols regulating blood serum cholesteroland esters thereof; diacylglycerol, γ-linolenic acid, α-linolenic acid,beet fiber, corn (maize) fiber, psyllium seed coat, tea polyphenol,lecithin; dried bonito peptide, sardine peptide, casein dodecapeptide,and soybean protein isolate which are effective in lowering bloodpressure; and lactic acid bacteria, gluconic acid, oligosaccharides, andvarious dietary fibers which improve the intestinal environment toregulate the intestines. Furthermore, foods or drugs with enhancedbioregulatory functions can be obtained by adding theβ-glucan-containing fat and oil composition of the present invention tosubstances known to have health improving functionality, such asChlorella, Spirulina, propolis, chitin, chitosan, nucleic acids, leyss(Ganoderma lucidum), agaricus, ginkgo leaf extract, lakanka (Lo Hon Go),turmeric, garcinia, apple fiber, gymnema, collagen, blueberry, aloe, sawpalmetto, plant fermentation enzymes, soybean isoflavon, chlorophyll,royal jelly, Asian ginseng, prune, and herbs, such as chamomile, thyme,sage, peppermint, lemon balm, mallow, oregano, cat nip tea, yarrow, andhibiscus.

When added to processed foods of rice, wheat, maize or soybeans, theβ-glucan-containing fat and oil composition of the present invention iscapable of imparting functionality to these foodstuffs or enhancing thefunctionality of the foodstuffs. Examples of such processed foods areboiled rice products (e.g., frozen boiled rice and sterile boiled rice);processed rice products, such as rice noodles, rice chips, and ricecrackers; the above-recited bakery products and confectionery products;noodles, such as pastas, buckwheat noodles, udon noodles, houtounoodles, and Chinese noodles; other wheat processed foods; breakfastcereals or processed maize products such as corn flakes; and processedsoybean products, such as tofu, soybean milk, soybean milk beverages,yuba (soybean milk skin), thin fried tofu, thick fried tofu, round friedtofu, soybean jam, and miso (soybean paste). Additionally, the fat andoil composition can be added to a variety of foods, including dairyproducts, such as milk, processed milk, yogurt, whey beverages,fermented lactic acid beverages, butter, and cheese; Japanese sweets,such as yokan (bean jelly), monaka (wafer with bean jam), and sweet beanjam; soups, such as potage, stew, and curry; seasonings, such as soysauce, Worcester sauce, dips, jams, and tomato ketchup; processed meatproducts, such as sausage; and processed marine products, such assteamed fish paste, baked fish paste and fried fish paste.

Among the known microorganisms suited for obtaining β-glucan are thoseof the genus Aureobasidium. The present invention provides novelmicroorganisms advantageous for the production of β-glucan. Thefollowing is the details of the novel microorganisms.

The microorganisms provided in the present invention are a microorganismof which the 18S rRNA gene contains the sequence of 1732 bases shown inSequence Listing, SEQ ID No. 1 or a base sequencemolecular-phylogenetically equivalent thereto based on the 18S rRNA genebase sequence and which is resistant to the antibiotic cycloheximideproduced by Streptomyces griseus, etc. and capable of secreting andproducing β-glucan out of fungi and a microorganism of which theITS-5.8S rRNA gene contains the sequence of 563 bases shown in SequenceListing, SEQ ID No. 2 or a base sequence molecular-phylogeneticallyequivalent thereto based on the ITS-5.8S rRNA gene base sequence andwhich is capable of secreting and producing β-glucan. The microorganismpreferably has resistance to the antibiotic cycloheximide produced byStreptomyces griseus, etc.

As long as the above-mentioned characteristics are possessed, anystrains can be used, including wild strains, stock strains, strainsdeposited with culture collections, mutants (induced by UV irradiationor treatment with chemicals, e.g., N-methyl-N′-nitrosoguanidine (NTG),acridine, ethane methane sulfonate (EMS), and nitrous acid), andimproved strains obtained through biological/genetic engineeringtechnology involving cell fusion, genetic modification, and the like.

The microorganisms of the present invention are obtained from theenvironment, for example, foods, vegetables, fruits, indoor and outdoorair (on settling plates), floors, walls, ceilings, roofs, mortar,concrete, tile joints, shower curtains, plastic cloth, refrigerators,washing machines, bathtubs, indoor dust, plant body surfaces, soil,river water, lake water, and sea water.

The present inventors have separated the microorganisms of the inventionfrom the environment as demonstrated in Examples given infra. Among theseparated microorganisms of the invention are found those belonging tothe genus Aureobasidium. In particular, a strain capable of secretingand producing non-colored, high-purity β-glucan out of fungi with goodefficiency was designated ADK-34. This strain was deposited withInternational Patent Organism Depositary, the National Institute ofAdvanced Industrial Science and Technology (address: Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, Japan; post code: 305-8566) on Jun. 2,2003 as labeled “Aureobasidium pullulans ADK-34” (labeling foridentification of microorganism), given accession number FERM BP-8391.

Drug resistance of a strain, namely the drug concentration at which astrain shows resistance can vary depending on the conditions of thestrain, the kind of the medium, and the cultivation time. The expression“resistant to cycloheximide” as used herein shall mean “exhibitingresistance to cycloheximide compared with reference strains, IFO-4466,IFO-6353, and IFO-7757”. This is the case that the formation of a colonyof 0.1 mm or greater, preferably 0.3 mm or greater, still preferably 0.5mm or greater in diameter as a result of fungous proliferation isobserved on a solid medium having the cycloheximide concentration thatIFO strains is not allowed to grow after a strain is inoculated on thesolid medium (agar plate) containing cycloheximide and cultured at 26°C. for 10 days. Usually, these IFO strains are not allowed to grow on asolid medium containing cycloheximide at concentrations of 20 μg/ml orhigher.

Whether a strain, a culture or β-glucan is “non-colored” or “withsuppressed coloration” as used herein is evaluated as follows. A cultureis diluted appropriately and centrifuged (10000×g, 10 mins) to removethe fungi. The absorbance of the resulting diluted culture solution at490 nm is measured. The strain, culture or β-glucan under test is called“non-colored” or “with suppressed coloration” when the absorbance thusmeasured is 0.099 or lower, preferably 0.060 or lower, still preferably0.050 or lower, under conditions such that a diluted culture solution ofIFO-6353 strain prepared in the same manner has an absorbance of 0.1 orhigher at 490 nm.

The term “high purity” as used in the invention is intended to have thefollowing meaning. In the measurement of purity with respect to theproduced polysaccharide pullulan as demonstrated in Analysis Example 3described infra, a phenol-sulfuric acid value of a test system ismeasured before and after treatment with pullulanase (enzyme). When theratio of the value after the enzyme treatment to that before the enzymetreatment is 75% or higher, the purity of the produced β-glucan is saidto be high. That ratio is preferably 85% or higher, still preferably 90%or higher.

The microorganisms of the present invention are suitable forextracellular production of β-glucan with high purity at highefficiency. The β-glucan produced by the microorganisms of the inventionis preferably one having at least a β-1,3-D-glucopyranose bond in itsstructure.

The process of producing β-glucan using the microorganisms of theinvention will be described with reference to its preferred embodiments.

According to the process, production of β-glucan using themicroorganisms of the invention is achieved by causing a strain of themicroorganism of the invention to act on a medium allowing the strain togrow to produce β-glucan out of fungi in the medium. The production ofβ-glucan out of fungi can also be achieved by separating culture fungiobtained by culturing a strain of the microorganism of the invention andcausing the culture fungi to act on a solution or medium containingsaccharides that are a substrate of β-glucan. There are cases whereinsecretive β-glucan that is ready to be secreted out of fungi is presentwithin the fungi. In the present invention, such β-glucan accumulated inthe fungi and ready to be secreted is also included under the term“secreted β-glucan out of fungi”.

The media in which a strain of the microorganisms of the invention iscultured in the process of producing β-glucan using the microorganismsof the invention include ordinary ones containing nutrients (includingcarbon sources, nitrogen sources, and inorganic salts) microorganismsbelonging to the genus Aureobasidium can usually utilize. The media mayfurther contain organic nutrients if needed. Liquid media containingsaccharides as a carbon source are preferred. Any of various syntheticmedia, semisynthetic media, natural media, and the like is usable.

The carbon sources preferably include saccharides. The saccharidesinclude monosaccharides, such as glucose, fructose, mannose, galactose,xylose, and arabinose; disaccharides, such as sucrose, maltose, lactose,and treharose; oligosaccharides, such as fructo-oligosaccharides andxylo-oligosaccharides; and polysaccharides, such as dextrin and starch.The saccharides can be used either individually or as a combinationthereof. It is still preferred to use, among them, hexoses, such asglucose and fructose; disaccharides, such as sucrose and lactose; orpolysaccharides, such as starch, dextrin, and hydrolyzates of suchcarbohydrates. Beet juice, sugarcane juice, fruit juices such as citrusjuice, or mixtures of these juices, either as such or sweetened, arealso useful. Additionally, other carbon sources including alcohols, suchas glycerol and ethylene glycol, sugar alcohols, such as mannitol,sorbitol, and erythritol, and organic acids are also used appropriately.These carbon sources may be added during cultivation as needed. Forexample, it is preferred that a saccharide such as sucrose beappropriately fed to the medium to keep its concentration within a rangeof from 3 to 500 g/l, still preferably 5 to 300 g/l, particularlypreferably 10 to 200 g/l, thereby to relatively increase the rate andamount of production of β-glucan.

The nitrogen sources include organic ones, such as peptone, meatextract, soybean flour, casein, amino acids, malt extract, corn steepliquor, casein decomposition products, yeast extract, and urea; andinorganic ones, such as sodium nitrate, ammonium sulfate, ammonia gas,and aqueous ammonia; and mixtures thereof.

The inorganic salts include sodium chloride, potassium chloride, calciumcarbonate, magnesium sulfate, sodium phosphate, potassium phosphate,cobalt chloride, and heavy metal salts. If desired, vitamins may beused. Where foaming occurs during cultivation, known defamers may beadded to the medium appropriately.

The cultivation conditions for the microorganism strains of theinvention are not particularly limited and can be chosen from the rangesallowing the strains to grow satisfactorily. Generally recommendedconditions are pH of 5.0 to 8.5, 20° to 35° C., and about 2 to 8 days.These cultivation conditions are subject to variation according to thekind and characteristics of the strain, external conditions, and soforth and can be selected to give the best results. The amount ofstrains to be inoculated into a medium is preferably one platinumloopful for flask cultivation. For increased scale production, a seedculture is added to a main culture in an amount of 1 to 10% (v/v) basedon the main culture. These amounts are not limitative as long ascultivation is possible.

The microorganism strains of the invention are cultivated under aerobicconditions with aeration-agitation, shaking, and the like. Cultivationis conducted until a desired β-glucan concentration is reached, usuallyfor 2 to 5 days. Beta-glucan production may be conducted in a continuousmanner by continuously feeding a saccharide, a β-glucan's substrate, orother medium components. The saccharide such as sucrose as a β-glucan'ssubstrate can be added in the form of powder or syrup (thick solution inwater) in an amount of 0.1 to 500 g per liter of the culture solution.Addition of more than 500 g/l results in great reduction of productionspeed. After addition of the substrate, the culture reaction is allowedto proceed preferably at 25° to 35° C. for 1 to 7 days, desirably about2 to 5 days, under aerobic conditions with shaking, aeration-agitationor like operation to produce β-glucan from the saccharide substrate,such as sucrose.

It is possible to use culture fungi obtained by cultivating themicroorganism strain of the invention or an extract of the culture fungias a catalyst for producing β-glucan. In this case, a solution or mediumcontaining a saccharide as a substrate is added to a suspension of theculture fungi, a suspension of the prepared culture fungi or asuspension of the treated culture fungi. The prepared culture fungiinclude a disrupted cell solution prepared by homogenizing the culturefungi. The treated culture fungi include immobilized fungi obtained byimmobilizing the culture fungi or the disrupted cell solution withalginate gel, ion-exchange resins, ceramics, chitosan, etc. Solutionsthat can be used to prepare the suspension of the culture fungi, theprepared or treated culture fungi include the above-described media andone of, or a mixture of one or more of, buffer solutions of tris-aceticacid, tris-hydrochloric acid, sodium succinate, sodium citrate, sodiumphosphate, potassium phosphate, etc. The pH of the buffer solutions ispreferably 3.5 to 9.0, still preferably 5.0 to 8.0, particularlypreferably 5.2 to 7.8. The culture fungi concentration in the suspensionis, while not limitative, suitably about 0.1% to 10% on a wet volumebasis.

The solution or medium containing saccharides as a substrate can beadded to the suspension in any amount at any concentration, but it ispreferred that the amount of the substrate added each time be within arange that maintains the activity of the fungi. For example, 0.1 to 500g of the substrate may be added all at once or in several dividedportions, or the substrate may be added continuously at a rate of about0.5 to 500 g/day, both per liter of the suspension. The solutioncontaining the fungi and the substrate is incubated at a giventemperature for a given period of time to produce β-glucan. Theincubation is preferably carried out under the same conditions as forthe cultivation of the aforementioned microorganism strains of theinvention.

After the cultivation, the secreted and produced β-glucan out of fungiis separated and harvested from the culture in a usual manner.Specifically, the solid matter, fungi etc., is separated and removedfrom the culture by centrifugation, filtration or like means, and theseparated culture solution may be purified by an appropriate combinationof known methods for removing impurities and salts, such as treatmentwith activated carbon, ion-exchange resins, etc. The thus harvestedβ-glucan may further be purified by any one of, or an appropriatecombination of, or a repetition of, additional purification methodsincluding adsorption-desorption using hydrophobic resins, solventprecipitation using ethanol, methanol, ethyl acetate, n-butanol, etc.,column or thin film chromatography using silica gel, etc., andpreparative high performance liquid chromatography using reverse phasecolumns.

The fungi may be sterilized either before or after separating thesecreted and produced β-glucan out of fungi by the above-describedmethods. The sterilization temperature is not particularly limited aslong as the fungi are killed but preferably 500 or higher, stillpreferably 60° C. or higher, particularly preferably 80° C. or higher.The temperature may be further raised, e.g., to 90° C. or higher, or at121° C. under pressure, whereby the secretive β-glucan accumulatedwithin the fungi and ready to be secreted out of fungi can be extractedwith hot water. While the sterilization time or the hot water extractiontime is arbitrarily selected, a period of from 10 minutes to 8 hours,preferably 15 minutes to 6 hours, particularly preferably 30 minutes to2 hours is suitable for controlling incorporation of impurity andpreventing deterioration of the β-glucan.

Beta-glucan can also be produced in the same manner as described above,except for replacing the microorganism according to the presentinvention with a microorganism of which the ITS-5.8S rRNA gene exhibitssequence homology of at least 98% with the base sequence shown inSequence Listing, SEQ ID No. 2.

It is preferred to use Aureobasidium pullulans ADK-34 (FERM BP-8391) forthe production of β-glucan because the resulting β-glucan has at least aβ-1,3-D-glucopyranose bond in the structure thereof.

The β-glucan production process using the microorganisms according tothe invention is advantageous over the processes using known strains ofAureobasidium pullulans or other microorganisms belonging to the genusAureobasidium in that the production of pullulan is significantlysuppressed. That is, the produced β-glucan can be isolated from theculture more easily, and the purification operation for recovering highpurity β-glucan can be simplified. Besides, the β-glucan produced byusing the microorganisms of the invention is less impure and remarkablyless colored than β-glucan extracted from the fungous cell walls ofother microorganisms of the genus Aureobasidium, yeasts, lactic acidbacteria, etc. or from basidiomycetes or plants. Therefore, the producedβ-glucan can be isolated and purified through simplified operations togive high and constant quality β-glucan in a stable manner with ease.

Being free from coloration and of high quality, the β-glucan obtained inthe present invention can be used in a wide variety of applications asthey are or as incorporated into other products.

Applications of the β-glucan obtained in the present invention includefoods, food additives, cosmetics, toiletries, chemical products, drugs,and so forth.

Specific examples of the foods are listed below.

Processed fat and oil foods include margarine, shortening, mayonnaise,cream, salad oil, frying oil, whipping cream, foamable emulsified fats,dressings, fat spreads, custard cream, and dipping cream. Because theβ-glucan obtained in the present invention has excellent compatibilitywith fats and oils as previously stated, it is preferred that theβ-glucan be added to fats and oils to give fat and oil compositionscontaining the β-glucan (i.e., the β-glucan-containing fat and oilcompositions according to the present invention), which are usedtogether with other raw materials.

Grain-related products include foods made mainly from wheat flour, foodsmade mainly from rice, processed rice products, processed wheatproducts, processed corn products, and processed soybean products.Examples are bakery products, such as bread, dessert bread, and Danishpies; pancakes, doughnuts, pizza, tempura, and premixes thereof;cookies/biscuits and snacks; noodles, such as raw noodles, dry noodles,packaged instant noodles, instant cup noodles, udon noodles, buckwheatnoodles, Chinese noodles, rice noodles, and pastas; and rice products,such as boiled rice, rice cake, sterile boiled rice, retort pouch boiledrice, nonglutinous rice flour, glutinous rice flour, dumplings, ricecrackers, and rice chips.

Confectionery products include Japanese or European ones, such aschocolates, candies, drops, chewing gums, baked confections, cakes, andsweet bean-jam buns.

Processed meat products include ham, sausage, and hamburgers. Processedmarine products include baked fish paste, steamed fish paste, fried fishpaste, and fish sausage.

Dairy products include butter, cheese, ice cream, and yogurt.

Beverages include alcoholic beverages, e.g., beer, sake, whisky, brandy,shochu, distilled liquors, sparkling alcohols, wines, and fruit wines;coffee, tea, green tea, woolong tea, Chinese tea, cocoa, carbonatedbeverages, nutritious drinks, sports drinks, coffee drinks, lactic acidbeverages, fruit juices, and fruit drinks.

Seasonings and sauces include spices, dips, dressings for meat, saladdressings, Worcester sauce, miso, soy sauce, and roux type sauce mixes,e.g., curry sauce mixes and hashed beef sauce mixes. Soups include cornsoup, potato soup, and pumpkin soup. Jams, peanut butter, and toppingsare also included in foods.

The foods also include preserved foods, such as canned or bottled foodsof fishes and shells, meats, fruits, vegetables, mushrooms, corned beef,jams, tomatoes, etc.; frozen foods; retort pouch foods, such as curry,stew, meat sauce, ma-po tofu, stew of meat with vegetables, soups, andboiled rice; and powdered foods, such as instant powder foods, e.g.,powdered beverages, powdered soups, and powdered miso soup.

The foods also include special health foods, medicated foods, nurseryfoods, such as baby foods, invalid diets (e.g., liquid diets), diets forthe old, fat-reducing diets, and supplementary foods.

Microwave foods ready to be reheated or cooked are also included infoods.

The food additives include emulsifiers, thickeners, thickeningstabilizers, food quality improvers, antioxidants, stabilizers,preservatives, flavors, sweeteners, colorants, bleaches, souring agents,gum bases, seasonings, bittering agents, nutrient enhancers, spices, andother non-categorized food additives.

The cosmetics and toiletries include skin care cosmetics, hair carecosmetics, medicated cosmetics, and oral care products. Examples includebasic skin care cosmetics, such as skin lotions, milky lotions, skinmilks, creams, ointments, lotions, calamine lotions, sun screens, suntanproducts, after-shave lotions, pre-shave lotions, makeup bases, beautymasks, facial cleansers, facial washes, antiacne cosmetics, andessences; makeup cosmetics, such as foundations, face powders, eyeshadows, eye liners, eyebrow colors, cheek colors, lipsticks, and nailcolors; shampoos, hair rinses, hair conditioners, hair dyes, hairtonics, waveset lotions, hair dressings, hair growth tonics, pilatories,body powders, deodorants, depilatories, soaps, body shampoos, handcleaners, perfumes, tooth pastes, oral care preparations, and bathingpreparations.

The chemical products include surface active agents, emulsifiers,thickeners, and viscosity modifiers.

The drugs include those having cholesterol reducing effect, intestinalregulatory effect, blood sugar controlling effect, and the like andtherefore effective in preventing habitual diseases, and drugs havingimmune enhancing action.

The present invention will now be illustrated in greater detail withrespect to Examples, but it should be understood that the invention isnot construed as being limited thereto. Unless otherwise noted, all theparts and percents are by weight.

ANALYSIS EXAMPLE 1 Confirmation of β-Glucan and Determination ofβ-Glucan Content

Analysis of β-glucan in polysaccharides was carried out by firstdetermining the total polysaccharide content that settled out of analcohol solution by the phenol-sulfuric acid method and then confirmingand determining β-glucan in the precipitated polysaccharides. Theconformation and determination of β-glucan were performed with an assaykit for detecting β-glucan having a β-1,3-D-glucopyranose bond,available from Seikagaku Corp. The analytical procedures are describedbelow.

The total polysaccharide content in a sample was measured by thephenol-sulfuric acid method as follows. To 30 μl of a sample solutionwas added 30 μl of distilled water, and 120 μL of a phosphate buffersolution containing 300 mM NaCl (pH 6.9) was added thereto. To themixture was added 540 μl (three times the volume) of ethanol, and thesystem was allowed to stand at −15° C. for 10 minutes to precipitatepolysaccharides. The supernatant liquid was removed, and 100 μl ofdistilled water was added to the residue to dissolve it. To the solutionwere added 100 μl of a 5 wt % aqueous phenol solution and 500 μl ofsulfuric acid and allowed to react. The absorbance of the reactionsolution at 490 nm was measured. A blank was prepared by adding 100 μlof a 5 wt % aqueous phenol solution and 500 μl of sulfuric acid to 100μl of distilled water containing no sample, and the absorbance of theblank at 490 nm was also measured. A two-fold dilution series wasprepared from 10 mg/ml pullulan as standard samples. A calibration curvewas prepared using the standard samples, and the total polysaccharidecontent in the sample under analysis was obtained from the calibrationcurve and the absorbances.

A sample solution having a total polysaccharide content of about 0.1 to1 mg/ml was 10-fold diluted with 1.0M NaOH and the 10¹⁰-fold dilutedwith β-glucan-free distilled water. Into a test tube was put 50 μl ofthe resulting β-glucan dilution, and 50 μl of the main reaction reagentof the kit was added, followed by incubation at 37° C. for 30 minutes.Subsequently, 50 μl of a prepared sodium nitrite solution, 50 μl ofammonium sulfamate, and 50 μl of a prepared N-methyl-2-pyrrolidonesolution were added to cause reaction. The absorbance of the reactionsolution at 545 nm (reference wavelength: 630 nm) was measured.Beta-glucan solutions having concentrations of from 7.5 to 60 pg/ml wereprepared using the attached β-glucan standard reagent to prepare acalibration curve. The concentration of the β-glucan dilution wascalculated from the calibration curve and the absorbance thereby toobtain the β-glucan content in the sample under analysis.

ANALYSIS EXAMPLE 2 Measurement of Molecular Weight of β-Glucan

The molecular weight of β-glucan was measured as follows. A sample wasmixed with three times as much alcohol, cooled to −20° C., and allowedto stand for 10 minutes to obtain a precipitate. A 5 mg portion of theprecipitated β-glucan was put into a test tube, and 1 ml of distilledwater was added thereto. The precipitate was dissolved in a boilingwater bath. The solution was filtered through a 0.22 μm filter toprepare a sample for HPLC. The sample was analyzed by HPLC using agel-filtration column, Shodex-packed column KS-805 (available from ShowaDenko K. K.) at a flow rate of 0.6 ml/min at a temperature of 50° C. AnR1 detector was used for detection, water was used as a developingsolvent, and Shodex Pullulan Standard β-82 (Showa Denko K. K.) was usedas a molecular weight marker.

ANALYSIS EXAMPLE 3 Measurement of Purity wrt Polysaccharide Pullulan inCulture

The purity with respect to polysaccharide pullulan produced in theculture was determined using pullulanase (available from Wako PureChemical Industries, Ltd.), an enzyme specifically decomposing pullulan.That is, the amount of the polysaccharides was measured before and afterthe enzymatic digestion with pullulanase, and the purity with respect tothe produced polysaccharide pullulan was calculated from the amount ofthe polysaccharides after the enzymatic treatment (i.e., the amount ofthe polysaccharides that had not been-digested with pullulanase) and thetotal amount of the polysaccharides before the enzymatic treatment. Thedetails of the polysaccharide determination are described below.

A mixture of 50 μl of a diluted pullulanase solution and 2 ml of acitrate buffer solution (containing 10 mmol/l citric acid and 20 mmol/lphosphate buffer; pH 6.0) was used as an enzyme solution. A dilutedsolution (10 mg/ml) of pullulan (available from Tokyo Kasei Kogyo Co.,Ltd.) was used as a standard sample solution positive control). Two 30μl portions of each of a culture solution under analysis and thestandard sample were put into the respective 1.5-ml volume test tubes.Into one of the tubes was added 30 μl of the enzyme solution, and 30 μlof PBS into the other, followed by allowing the systems to react at 37°C. for 1 hour. After the reaction, three times as much ethanol as thecontents was added to each tube, and the contents were stirred well andincubated at −15° C. for 10 minutes, followed by centrifugal separationat 4° C. and 15000 rpm for 10 minutes. The supernatant liquid wasdiscarded, and the residue was dried. To the resultant solid was added100 μl of distilled water, the mixture stirred, and 100 μl of a 5 wt %aqueous phenol solution added, and 500 μl of sulfuric acid addedimmediately, to cause color reaction. After cooling, the reactionsolution was dispensed in 100 μl portions into a 96-well microplate. Theabsorbance at 490 nm was measured. A mixture of distilled water, a 5 wt% aqueous phenol solution, and sulfuric acid was used as a blank. Acalibration curve was prepared using pullulan solutions diluted from 10mg/ml to 1 μg/ml concentrations. The polysaccharide contents in theculture solution before and after the enzymatic digestion were obtainedfrom the calibration curve and the absorbances, from which the puritywith respect to the polysaccharide pullulan in the culture solution wascalculated. When the standard sample (pullulan solution) wasenzymatically digested, the digestion rate achieved was 90% or higher ata concentration of 10 mg/ml, 95% or higher at 1 mg/ml, and 98% or higherat 0.1 mg/ml or lower concentrations. Thus, where pullulan had beenproduced, the phenol-sulfuric acid value (absorbance) decreased afterthe treatment with the enzyme pullulanase. In case when, for example,the absorbance decreased by 50%, the purity with respect to thepolysaccharide pullulan was calculated to be 50%.

PREPARATION EXAMPLE 1 Preparation of β-Glucan of Mushroom Origin

1) Preparation of Mushroom Extract

The hymenia of Agaricus blazei were ground. To 10 kg of the groundmaterial was added 50 liters of hot water. The suspension was gentlystirred while boiling for 3 hours to conduct hot water extraction,followed by centrifugal separation to obtain an extract.

2) Purification of Mushroom Extract

To the extract obtained in (1) above was added three times as much 99%ethyl alcohol. The thus formed precipitate was collected and lyophilizedto give 1200 g of a crude product (crude β-glucan of mushroom origin,designated sample A). The β-glucan content per gram of sample A wascalculated to 860 mg. The molecular weight at the peak was 1,000,000.

3) Preparation of Enzymatically Treated Mushroom Extract

One kilogram of the hymenia of Agaricus blazei was ground in a mixerwith 2 liters of water. Two grams of Funcelase (from YakultPharmaceutical Ind. Co., Ltd.) was added thereto and mixed, followed byincubation at 55° C. for 3 hours for enzymatic reaction. The reactionsystem was heated to 85° C. and maintained at that temperature for 10minutes to deactivate the enzyme. Three liters of distilled water wasadded, and the mixture was stirred well. The solid matter was removed togive 4.5 liters of an extract (enzymatically treated β-glucan extract ofmushroom origin, designated sample B). The β-glucan content permilliliter of sample B was calculated to be 93 mg. The molecular weightswere distributed between 10,000 and 800,000, with the peak molecularweight being 120,000.

4) Preparation of Mushroom Mycelium Culture

Into each of four 500 ml volume Erlenmeyer flasks was put 120 ml of aglucose-potato extract medium (glucose 2%; potato 200 g/l) andsterilized at 120° C. for 30 minutes. The hyphae of Flammulina velutipesIFO-30602 separately maintained on a slant culture medium wereinoculated into the medium and cultured in a rotary incubator at 25° C.and 200 rpm for 10 days. The cultures from the four flasks werecombined, washed with physiological saline, and lyophilized to obtain 8g of dry mycelia. One gram of the resulting mycelia was extracted with10 ml of a 0.2M aqueous sodium hydroxide solution at 15° C. for one daywith stirring. The extraction system as such was adjusted to pH 3.0 withhydrochloric acid, autoclaved at 120° C. for 30 minutes, andcentrifuged. The separated supernatant liquid was adjusted to pH 7.0with disodium phosphate, and three times as much ethyl alcohol wasadded. The thus formed precipitate was collected, and 10 ml of distilledwater was added thereto to obtain sample C. The β-glucan content insample C was calculated to be 40 mg per milliliter. The molecular weightat the peak was 200,000.

PREPARATION EXAMPLE 2 Preparation of β-Glucan of Microorganism Origin

1) Preparation of Microbial Cell Wall

Fungi were suspended at a concentration of 1 g/ml in water having 0.5%lysolecithin dissolved therein. The suspension was treated on anultrasonic disrupter for 10 minutes, followed by centrifugal separationto remove the supernatant liquid. The solid was lyophilized to obtain acell wall component. The cell wall obtained from a commerciallyavailable powdered lactic acid bacterium (from Morinaga Milk IndustryCo., Ltd.) was designated sample D. The cell wall obtained fromcommercially available compressed yeast (Dia Yeast from Kyowa HakkoKogyo Co., Ltd.) was designated sample E. The cell wall obtained fromcommercially available dry chlorella (Chlorella Micropowder fromChlorella Kogyo Co., Ltd.) was designated sample F. Ten milligrams ofeach sample was extracted with 1 ml of 1M sodium hydroxide aqueoussolution at 50° C. for one day. The supernatant liquid (extract)separated by centrifugation was diluted with distilled water to preparea 10-fold to 100-fold dilution series to carry out β-glucan contentdetermination. As a result, the β-glucan content per 10 mg of Samples D,E, and F was found to be 2.8 mg, 4.9 mg, and 3.5 mg, respectively. Themolecular weight was then measured. Ten milligrams of each sample wasextracted with 1 ml of 1M sodium hydroxide aqueous solution at 50° C.for one day, followed by centrifugation. To the separated supernatantliquid was added three times as much ethanol. The precipitate thusformed was dissolved in 1 ml of distilled water. Samples D, E, and Fwere found to have a weight average molecular weight of 1,200,000,2,000,000, and 1,800,000, respectively.

PREPARATION EXAMPLE 3 Preparation of Alkali Extract From Cell Walls

1) Preparation of Alkali Extract From Fungal Cell Walls

A hundred grams of Sparassis crispa was extracted with 1 liter of 1%sodium hydroxide at 65° C. for 2 hours with stirring. The extractionresidue was removed by centrifugation. The extract was neutralized withHCl, and an equivalent amount of ethanol was added thereto to recover 20g of a precipitate (β-glucan extracted with fungal cell walls,designated sample G). The β-glucan content per gram of sample G wascalculated to be 500 mg. The peak molecular weight was 1,200,000.

2) Preparation of Alkali Extract From Microbial Cell Walls

A hundred grams of cell walls prepared from commercially availablecompressed yeast (sample E) was extracted with 1 liter of 2% sodiumhydroxide at 4° C. for 24 hours. The extract solution separated bycentrifugation was neutralized with HCl, and double the amount ofethanol was added thereto to collect 20 g of a solid extract (β-glucanextracted from yeast cell walls, designated sample H). The β-glucancontent per 10 grams of sample H was calculated to be 4.2 mg. The peakmolecular weight was 1,600,000.

PREPARATION EXAMPLE 4 Preparation of Microbial Culture

1) Preparation of Lactic Acid Bacterium Culture

A culture medium was prepared by mixing, in ratio, 5 g of polypepton, 5g of yeast extract, 5 g of glucose, 1 g of MgSO₄.7H₂O, and 1 liter ofdistilled water and adjusting to pH 5.5. Pediococcus damnosus (IFO-3896at Institute for Fermentation, Osaka) was inoculated into 5 liters ofthe medium and cultured at 30° C. for 5 days with agitation (50 rpm) andwithout aeration in duplicate. The combined culture (10 liters) wascentrifuged, and the separated supernatant liquid was concentrated to 2liters under reduced pressure. To the concentrate was added double thevolume of ethanol, and the formed precipitate was collected andlyophilized to give 15 g of a lactic acid culture as powder (designatedsample I). The β-glucan content per gram of sample I was calculated tobe 600 mg. The peak molecular weight was 1,900,000.

2) Preparation of Aureobasidium Culture

Aureobasidium pullulans IFO-7757, which is a genetically andmorphologically identified microorganism belonging to the genusAureobasidium and capable of producing a glucose polymer having a β-bondout of fungi, cultured on a potato-dextrose-agar slant was used as astock strain. The strain was inoculated into 100 ml of YM liquid medium(from Difco) in a 500 ml volume Erlenmeyer flask and pre-cultured at 28°C. for 3 days. The resulting preculture was put into a 5 liter volumefermentation tank equipped with impellers “FULLZONE” containing 3 litersof Czapek's medium (from Difco) and cultured at 28° C. for 5 days. 1During the culturing, the medium was adjusted at pH 5.0, and theaeration rate was set at 1 vvm by sequentially controlling the amount ofair and the number of revolution. The culture (3 liters) was sterilizedby heating at 90° C. for 30 minutes and centrifuged to remove the fungi.One liter of the culture solution was lyophilized to give 27 g of afreeze-dried Aureobasidium culture, designated sample J. The β-glucancontent per gram of sample J was calculated to be 440 mg. To a 10 mg/mlsolution of sample J in distilled water was added a suspension of enzymepullulanase (from Wako Pure Chemical) to give an enzyme concentration of0.05%. After allowing the system to react for 2 hours, double thequantity of ethanol was added thereto. The thus precipitated solid wasre-dissolved in 1 ml of distilled water to measure the molecular weight.For control, a 10 mg/ml solution of pullulan in distilled water wastreated in the same manner to measure the molecular weight. Sample J hada peak molecular weight of 200,000, whereas the pullulan solution showedno molecular peak.

To the rest of the culture (2 liters) was added double the volume ofethanol, and the formed precipitate was collected and freeze-dried togive 26 g of a purified Aureobasidium culture as powder, designatedsample K. The β-glucan content per 10 milligrams of sample K wascalculated to be 6 mg. To a 10 mg/ml solution of sample K in distilledwater was added a suspension of enzyme pullulanase (from Wako PureChemical) to give an enzyme concentration of 0.05%. After allowing thesystem to react for 2 hours, double the quantity of ethanol was addedthereto. The thus precipitated solid was re-dissolved in 1 ml ofdistilled water to measure the molecular weight. For control, a 10 mg/mlsolution of pullulan in distilled water was treated in the same mannerto measure the molecular weight. Sample K had a peak molecular weight of200,000, whereas the pullulan solution showed no molecular peak.

EXAMPLE 1 Beta-Glucan-Containing Fat and Oil Composition

A hundred parts of sample A and 100 parts of soybean oil were thoroughlymixed in a kneader. The mixture was allowed to stand at 60° C. for 10minutes and then cooled to room temperature, whereupon it became creamyto give a β-glucan-containing fat and oil composition-i according to thepresent invention (β-glucan content: 43%). The β-glucan was founduniformly dispersed in the composition.

EXAMPLE 2 Beta-Glucan-Containing Fat and Oil Composition

Eighty parts of sample B and 120 parts of soybean oil were thoroughlymixed in a kneader. The mixture was allowed to stand at 60° C. for 10minutes and then cooled to room temperature, whereupon it became creamyto give a β-glucan-containing fat and oil composition-2 according to thepresent invention (β-glucan content: 3.7%). The β-glucan was founduniformly dispersed in the composition.

EXAMPLE 3 Beta-Glucan-Containing Fat and Oil Composition

A hundred parts of sample H and 100 parts of soybean oil were thoroughlymixed in a kneader. The mixture was allowed to stand at 60° C. for 10minutes and then cooled to room temperature, whereupon it became creamyto give a β-glucan-containing fat and oil composition-3 according to thepresent invention (β-glucan content: 21%). The β-glucan was founduniformly dispersed in the composition.

EXAMPLE 4 Beta-Glucan-Containing Fat and Oil Composition

To three hundred parts of sample K were added 100 parts of palm oilhaving been melted at 70° C. and 1 part of lecithin, followed by mixingin a high-speed homomixer. The mixture was left to stand at 50° C. for20 minutes and then cooled to room temperature to give a lumpyβ-glucan-containing fat and oil composition-4 according to the presentinvention (β-glucan content: 44.9%). The β-glucan was found uniformlydispersed in the composition.

EXAMPLE 5 Beta-Glucan-Containing Fat and Oil Composition

To three hundred parts of sample E were added 100 parts of palm oilhaving been melted at 70° C. and 1 part of lecithin, followed by mixingin a high-speed homomixer. The mixture was left to stand at 50° C. for20 minutes and then cooled to room temperature to give a lumpyβ-glucan-containing fat and oil composition-S according to the presentinvention (β-glucan content: 36.6%). The β-glucan was found uniformlydispersed.

EXAMPLE 6 Beta-Glucan-Containing Fat and Oil Composition

To three hundred parts of sample F were added 100 parts of palm oilhaving been melted at 70° C. and 1 part of lecithin, followed by mixingin a high-speed homomixer. The mixture was left to stand at 50° C. for20 minutes and then cooled to room temperature to give a lumpyβ-glucan-containing fat and oil composition-6 according to the presentinvention (β-glucan content: 26%). The β-glucan was found uniformlydispersed.

EXAMPLE 7 Beta-Glucan-Containing Fat and Oil Composition

To fifty parts of sample G were added 30 parts of palm olein oil, 70parts of rapeseed oil, and 0.2 parts of protease-hydrolyzed egg yolk,followed by mixing in a mixer. The mixture was left to stand at 65° C.for 15 minutes and then cooled to room temperature to give a creamyβ-glucan-containing fat and oil composition-7 according to the presentinvention (β-glucan content: 16.6%). The β-glucan was found uniformlydispersed.

EXAMPLE 8 Beta-Glucan-Containing Fat and Oil Composition

To fifty parts of sample I were added 30 parts of palm olein oil, 70parts of rapeseed oil, and 0.2 parts of protease-hydrolyzed egg yolk,followed by mixing in a mixer. The mixture was allowed to stand at 65°C. for 15 minutes and then cooled to room temperature to give a creamyβ-glucan-containing fat and oil composition-8 according to the presentinvention (β-glucan content: 20%). The β-glucan was found uniformlydispersed in the composition.

EXAMPLE 9 Beta-Glucan-Containing Fat and Oil Composition

To fifty parts of sample K were added 30 parts of palm olein oil, 70parts of rapeseed oil, and 0.2 parts of protease-hydrolyzed egg yolk,followed by mixing in a mixer. The mixture was allowed to stand at 65°C. for 15 minutes and then cooled to room temperature to give a creamyβ-glucan-containing fat and oil composition-9 according to the presentinvention (β-glucan content: 20%). The β-glucan was found uniformlydispersed.

EXAMPLE 10 Beta-Glucan-Containing Fat and Oil Composition

To five parts of sample A were added 40 parts of rice oil, 20 parts ofolive oil, and 35 parts of safflower oil, followed by mixing in ahigh-speed homomixer. The resulting mixture was allowed to stand at 50°C. for 30 minutes and then cooled to room temperature to give aβ-glucan-containing fat and oil composition-10 according to the presentinvention (β-glucan content: 4.3%), which had almost the same viscosityas the raw material oils but showed slight turbidity. The β-glucan wasfound uniformly dispersed in the composition.

EXAMPLE 11 Beta-Glucan-Containing Fat and Oil Composition

To five parts of sample I were added 40 parts of rice oil, 20 parts ofolive oil, and 35 parts of safflower oil, followed by mixing in ahigh-speed homomixer. The resulting mixture was allowed to stand at 50°C. for 30 minutes and then cooled to room temperature to give aβ-glucan-containing fat and oil composition-11 according to the presentinvention (β-glucan content: 3%), which had almost the same viscosity asthe raw material oils but showed slight turbidity. The β-glucan wasfound uniformly dispersed.

EXAMPLE 12 Beta-Glucan-Containing Fat and Oil Composition

To five parts of sample J were added 40 parts of rice oil, 20 parts ofolive oil, and 35 parts of safflower oil, followed by mixing in ahigh-speed homomixer. The resulting mixture was allowed to stand at 50°C. for 30 minutes and then cooled to room temperature to give aβ-glucan-containing fat and oil composition-12 according to the presentinvention (β-glucan content: 2.2%), which had almost the same viscosityas the raw material oils but showed slight turbidity. The β-glucan wasfound uniformly dispersed.

EXAMPLE 13 Beta-Glucan-Containing Fat and Oil Composition

To thirteen parts of sample G were added 20 parts of hardened soybeanoil (melting point: 45° C.), 35 parts of palm oil, 30 parts of cottonseed oil, and 0.2 parts of soybean lysolecithin. After being allowed tostand at 70° C. for 10 minutes, the mixture was emulsified in ahigh-speed mixer and rapidly cooled for plasticization to give aβ-glucan-containing fat and oil composition-13 according to the presentinvention having margarine-like physical properties (β-glucan content:6.6%). The β-glucan was found uniformly dispersed in the composition.

EXAMPLE 14 Beta-Glucan-Containing Fat and Oil Composition

To thirteen parts of sample H were added 20 parts of hardened soybeanoil (melting point: 45° C.), 35 parts of palm oil, 30 parts of cottonseed oil, and 0.2 parts of soybean lysolecithin. After being allowed tostand at 70° C. for 10 minutes, the mixture was emulsified in ahigh-speed mixer and rapidly cooled for plasticization to give aβ-glucan-containing fat and oil composition-14 according to the presentinvention having margarine-like physical properties (β-glucan content:5.6%). The β-glucan was found uniformly dispersed.

EXAMPLE 15 Beta-Glucan-Containing Fat and Oil Composition

To thirteen parts of sample K were added 20 parts of hardened soybeanoil (melting point: 45° C.), 35 parts of palm oil, 30 parts of cottonseed oil, and 0.2 parts of soybean lysolecithin. After being allowed tostand at 70° C. for 10 minutes, the mixture was emulsified in ahigh-speed mixer and rapidly cooled for plasticization to give aβ-glucan-containing fat and oil composition-15 according to the presentinvention having margarine-like physical properties (β-glucan content:8%). The β-glucan was found uniformly dispersed.

EXAMPLE 16 Beta-Glucan-Containing Fat and Oil Composition

To fifty parts of sample B were added 27.6 parts of hardened fish oil(melting point: 36° C.), 18 parts of corn salad oil, and 0.4 parts ofglycerol monotartrate. The mixture was stirred at 50° C. for 30 minutes,emulsified in a high-speed mixer, and rapidly cooled for plasticizationto give a β-glucan-containing fat and oil composition-16 according tothe present invention having fat spread-like physical properties(β-glucan content: 4.8%). The β-glucan was found uniformly dispersed.

EXAMPLE 17 Beta-Glucan-Containing Fat and Oil Composition

To fifty parts of sample H were added 27.6 parts of hardened fish oil(melting point: 36° C.), 18 parts of corn salad oil, and 0.4 parts ofglycerol monotartrate. The mixture was stirred at 50° C. for 30 minutes,emulsified in a high-speed mixer, and rapidly cooled for plasticizationto give a β-glucan-containing fat and oil composition-17 according tothe present invention having fat spread-like physical properties(β-glucan content: 22%). The β-glucan was found uniformly dispersed.

EXAMPLE 18 Beta-Glucan-Containing Fat and Oil Composition

To fifty parts of sample I were added 27.6 parts of hardened fish oil(melting point: 36° C.), 18 parts of corn salad oil, and 0.4 parts ofglycerol monotartrate. The mixture was stirred at 50° C. for 30 minutes,emulsified in a high-speed mixer, and rapidly cooled for plasticizationto give a β-glucan-containing fat and oil composition-18 according tothe present invention having fat spread-like physical properties(β-glucan content: 31%). The β-glucan was found uniformly dispersed.

EXAMPLE 19 Beta-Glucan-Containing Fat and Oil Composition

To twenty parts of sample G were added 0.3 parts of olive oil (meltingpoint: 36° C.) and 0.1 parts of sodium casein. The mixture was left tostand at 55° C. for 15 minutes, emulsified in a high-speed mixer, andspray-dried to give a β-glucan-containing fat and oil composition-19according to the present invention in powder form (β-glucan content:49%). The β-glucan was found uniformly dispersed.

EXAMPLE 20 Beta-Glucan-Containing Fat and Oil Composition

To twenty parts of sample H were added 0.3 parts of olive oil (meltingpoint: 36° C.) and 0.1 parts of sodium casein. The mixture was allowedto stand at 55° C. for 15 minutes, emulsified in a high-speed mixer, andspray-dried to give a β-glucan-containing fat and oil composition-20according to the present invention in powder form (β-glucan content:41%). The β-glucan was found uniformly dispersed.

EXAMPLE 21 Beta-Glucan-Containing Fat and Oil Composition

To twenty parts of sample K were added 0.3 parts of olive oil (meltingpoint: 36° C.) and 0.1 parts of sodium casein. The mixture was allowedto stand at 55° C. for 15 minutes, emulsified in a high-speed mixer, andspray-dried to give a β-glucan-containing fat and oil composition-21according to the present invention in powder form (β-glucan content:59%). The β-glucan was found uniformly dispersed.

EXAMPLE 22 Production of Shortening

An oil phase consisting of 30 parts of palm oil, 50 parts ofhydrogenated palm oil, 20 parts of rapeseed oil, and 0.3 parts oflecithin was melted at 70° C. To 100 parts of the oil phase was added5.0 parts of sample G, and the mixture was allowed to stand at 70° C.for 30 minutes. The mixture was agitated in a homomixer at a highrotational speed for 2 minutes to prepare a β-glucan-containing fat andoil composition-22 according to the present invention. The β-glucan wasfound by visual observation sufficiently dispersed in the fat and oilphase. The composition was rapidly cooled for plasticization and cooledto 5° C. to obtain shortening (β-glucan content: 2.4%) according to thepresent invention. The resulting shortening was evaluated for smoothnessand flavor. The results obtained are shown in Table 1. It is understoodthat the resulting shortening is superior to that of Comparative Example1 described later in smoothness and flavor. Although the step of crystalaging had been omitted, the shortening can be said to enjoy the effectsof forming moderate crystals with excellent texture, accelerating suchcrystallization, and minimizing reduction of flavor by the emulsifier.

EXAMPLE 23 Production of Shortening

An oil phase consisting of 30 parts of palm oil, 50 parts ofhydrogenated palm oil, 20 parts of rapeseed oil, and 0.3 parts oflecithin was melted at 700C. To 100 parts of the oil phase was added 5.0parts of sample K, and the mixture was allowed to stand at 70° C. for 30minutes. The mixture was then agitated in a homomixer at a highrotational speed for 2 minutes to give a β-glucan-containing fat and oilcomposition-23 according to the present invention. The β-glucan wasfound by visual observation thoroughly dispersed in the fat and oil. Thecomposition was rapidly cooled for plasticization and cooled to 5° C. toobtain shortening (β-glucan content: 2.9%) according to the presentinvention. The resulting shortening was evaluated for smoothness andflavor. The results obtained are shown in Table 1. It is seen that theresulting shortening is superior to that of Comparative Example 1described below in smoothness and flavor. Although the step of crystalaging had been omitted, the shortening can be said to enjoy the effectsof forming moderate crystals with excellent texture, accelerating suchcrystallization, and minimizing reduction of flavor by the emulsifier.

COMPARATIVE EXAMPLE 1 Production of Comparative Shortening

An oil phase consisting of 30 parts of palm oil, 50 parts ofhydrogenated palm oil, 20 parts of rapeseed oil, and 0.3 parts oflecithin was melted at 70° C., stirred in a homomixer at a highrotational speed for 2 minutes, rapidly cooled for plasticization, andcooled to 5° C. to obtain shortening. The resulting shortening wasevaluated for smoothness and flavor. The results obtained are shown inTable 2, which prove the resulting shortening to be much inferior inflavor.

EXAMPLE 24 Production of Margarine

A hundred parts of an edible fat and oil consisting of palm oil,hydrogenated palm oil, rapeseed oil, and a sorbitan fatty acid ester ata weight ratio of 30:50:20:0.3 was melted at 70° C. Eight parts ofsample G was added thereto, and the mixture was left to stand at 65° C.for 30 minutes. A solution of 0.5 parts of defatted milk powder and 1part of edible salt in 16 parts of hot water (70° C.) was slowly addedto the mixture and mixed therewith while stirring in a homomixer. Then,the mixture was rapidly cooled for plasticization, maintained at 25° C.overnight, and cooled to 5° C. to give margarine according to thepresent invention (β-glucan content: 3.2%). The β-glucan was founduniformly dispersed. The resulting margarine was evaluated forstability, smoothness, and flavor. The results are shown in Table 1. Theresulting margarine had a fine and smooth texture. Besides, themargarine was more flavorful than that of Comparative Example 2described later, proving the effect of suppressing a reduction in flavorby the emulsifier.

EXAMPLE 25 Production of Margarine

A hundred parts of an edible fat and oil consisting of palm oil,hydrogenated palm oil, rapeseed oil, and a sorbitan fatty acid ester ata weight ratio of 30:50:20:0.3 was melted at 70° C. Eight parts ofsample H was added thereto, and the mixture was left to stand at 65° C.for 30 minutes. A solution of 0.5 parts of defatted milk powder and 1part of edible salt in 16 parts of hot water (70° C.) was slowly addedto the mixture and mixed therewith while stirring in a homomixer. Then,the mixture was rapidly cooled for plasticization, maintained at 25° C.overnight, and cooled to 5° C. to give margarine according to thepresent invention (β-glucan content: 2.7%). The β-glucan was founduniformly dispersed. The resulting margarine was evaluated forstability, smoothness, and flavor. The results are shown in Table 1. Theresulting margarine had a fine and smooth texture. Besides, themargarine was more flavorful than that of Comparative Example 2described later, proving the effect of suppressing a reduction in flavorby the emulsifier.

EXAMPLE 26 Production of Margarine

A hundred parts of an edible fat and oil consisting of palm oil,hydrogenated palm oil, rapeseed oil, and a sorbitan fatty acid ester ata weight ratio of 30:50:20:0.3 was melted at 70° C. Eight parts ofsample K was added thereto, and the mixture was left to stand at 65° C.for 30 minutes. A solution of 0.5 parts of defatted milk powder and 1part of edible salt in 16 parts of hot water (70° C.) was slowly addedto the mixture and mixed therewith while stirring in a homomixer. Then,the mixture was rapidly cooled for plasticization, maintained at 25° C.overnight, and cooled to 5° C. to give margarine according to thepresent invention (β-glucan content: 3.8%). The β-glucan was founduniformly dispersed. The resulting margarine was evaluated forstability, smoothness, and flavor. The results are shown in Table 1. Theresulting margarine had a fine and smooth texture. Besides, themargarine was more flavorful than that of Comparative Example 2 below,proving the effect of suppressing a reduction in flavor by theemulsifier.

COMPARATIVE EXAMPLE 2 Production of Comparative Margarine

A hundred parts of an edible fat and oil consisting of palm oil,hydrogenated palm oil, rapeseed oil, and a sorbitan fatty acid ester ata weight ratio of 30:50:20:0.3 was melted at 70° C. A solution of 0.5parts of defatted milk powder and 1 part of edible salt in 16 parts ofhot water (70° C.) was slowly added to the mixture and mixed therewithwhile stirring in a homomixer. Then, the mixture was rapidly cooled forplasticization, maintained at 25° C. overnight and cooled to 5° C. toobtain margarine. The resulting margarine was evaluated for stability,smoothness, and flavor. The results are shown in Table 2.

EXAMPLE 27 Production of Dressing

Ten parts of sample G, 10 parts of egg yolk, 1.5 parts of edible salt,11 parts of vinegar, 2.5 parts of soft white sugar, 0.05 parts ofmustard powder, and 0.05 parts of onion powder were mixed by agitatingin a mixer at a high speed for 5 minutes to prepare an aqueous phase.While the aqueous phase was further agitated in a homomixer at a highspeed, 75 parts of soybean salad oil heated to 70° C. was slowly addedthereto and mixed. The mixture was left to stand at 50° C. for 10minutes, emulsified, and cooled at 5° C. for 24 hours to obtain adressing (β-glucan content: 4.5%) according to the present invention.The β-glucan was found uniformly dispersed. The resulting dressing wasevaluated for stability and flavor. The results are shown in Table 1.The resulting dressing was proved excellent in stability and flavor.

COMPARATIVE EXAMPLE 3 Production of Comparative Dressing

Ten parts of egg yolk, 1.5 parts of edible salt, 11 parts of vinegar,2.5 parts of soft white sugar, 0.05 parts of mustard powder, and 0.05parts of onion powder were mixed by stirring in a mixer at a high speedfor 5 minutes to prepare an aqueous phase. A dressing was prepared byusing the aqueous phase in the same manner as in Example 27. Theresulting dressing was evaluated for stability and flavor. The resultsare shown in Table 2.

EXAMPLE 28 Production of Dressing

Ten parts of egg yolk, 1.5 parts of edible salt, 11 parts of vinegar,2.5 parts of soft white sugar, 0.05 parts of mustard powder, and 0.05parts of onion powder were mixed by agitating in a mixer at a high speedfor 5 minutes to prepare an aqueous phase. While the aqueous phase wasfurther agitated in a homomixer at a high speed, 75 parts of theβ-glucan-containing fat and oil composition-12 of Example 12 was slowlyadded thereto and mixed. The mixture was emulsified and cooled at 5° C.for 24 hours to obtain a dressing (β-glucan content: 1.65%) according tothe present invention. The β-glucan was found uniformly dispersed. Theresulting dressing was evaluated for stability and flavor. The resultsare shown in Table 1. The resulting dressing was proved excellent instability and flavor.

COMPARATIVE EXAMPLE 4 Production of Comparative Dressing

Ten parts of egg yolk, 1.5 parts of edible salt, 11 parts of vinegar,2.5 parts of soft white sugar, 0.05 parts of mustard powder, and 0.05parts of onion powder were mixed by agitating in a mixer at a high speedfor 5 minutes to prepare an aqueous phase. While the aqueous phase wasfurther agitated in a homomixer at a high speed, 75 parts of mixed fatsand oils (40 parts of rice oil, 20 parts of olive oil, and 35 parts ofsafflower oil) was slowly added thereto and mixed. The mixture wasemulsified and cooled at 5° C. for 24 hours to obtain a dressing. Theresulting dressing was evaluated for stability and flavor. The resultsare shown in Table 2.

EXAMPLE 29 Production of Mayonnaise

Thirty parts of soybean salad oil was added to 30 parts of sample H, andthe mixture was agitated for preliminary emulsification to prepare aβ-glucan-containing fat and oil composition according to the presentinvention. A thoroughly agitated mixture consisting of 9 parts of eggyolk, 5.2 parts of starch, 8.2 parts of sugar, 2.8 parts of edible salt,8 parts 10 of vinegar, 1 part of seasoning spices, and 6 parts of waterwas added to the fat and oil composition, and the mixture was emulsifiedin a colloid mill to make mayonnaise (β-glucan content: 12.6%) accordingto the present invention. The β-glucan was found uniformly dispersed.The mayonnaise was evaluated for stability, smoothness, and flavor. Theresults obtained are shown in Table 1. The resulting mayonnaiseunderwent no phase separation of water when stored for 1 month and had asmooth texture and a very good flavor.

COMPARATIVE EXAMPLE 5 Production of Comparative Mayonnaise

Thirty parts of soybean salad oil was added to 30 parts of water, andthe mixture was agitated for preliminary emulsification to prepare a fatand oil composition. A thoroughly stirred mixture consisting of 9 partsof egg yolk, 5.2 parts of starch, 8.2 parts of sugar, 2.8 parts ofedible salt, 8 parts of vinegar, 1 part of seasoning spices, and 6 partsof water was added to the fat and oil composition, and the mixture wasemulsified in a colloid mill to prepare mayonnaise, which was evaluatedfor stability, smoothness and flavor. The results obtained are shown inTable 2.

EXAMPLE 30 Production of Mayonnaise

Nine parts of egg yolk, 8.2 parts of sugar, 2.8 parts of edible salt, 8parts of vinegar, 1 part of seasoning spices, and 36 parts of sample Bwere mixed to prepare an aqueous phase. To the aqueous phase were added25 parts of rapeseed oil and 10 parts of the β-glucan-containing fat andoil composition-3 of Example 3. The mixture was preliminarily emulsifiedby agitating and then further emulsified in a colloid mill to obtainmayonnaise (β-glucan content: 5.5%) according to the present invention.The β-glucan was found uniformly dispersed. The resulting mayonnaise wasevaluated for stability, smoothness, and flavor. The results are shownin Table 1. The resulting mayonnaise underwent no separation of waterwhen stored for 1 month and had a smooth texture and a very good flavor.

COMPARATIVE EXAMPLE 6 Production of Comparative Mayonnaise

Nine parts of egg yolk, 8.2 parts of sugar, 2.8 parts of edible salt, 8parts of vinegar, 1 part of seasoning spices, and 36 parts of water weremixed to prepare an aqueous phase. To the aqueous phase were added 25parts of rapeseed oil and 10 parts of palm oil. The mixture waspreliminarily emulsified by agitating and then further emulsified in acolloid mill to obtain mayonnaise. The resulting mayonnaise wasevaluated for stability, smoothness, and flavor. The results are shownin Table 2.

EXAMPLE 31 Production of Fat Spread

A mixture consisting of 27.6 parts of hydrogenated fish oil (meltingpoint: 36° C.), 18.4 parts of cotton seed oil, 40 parts of sample K,12.3 parts of water, 1 part of edible salt, 0.5 parts of defatted milkpowder, 0.2 parts of flavor, and 0.3 parts of lecithin was emulsifiedand rapidly cooled for plasticization to prepare fat spread of thepresent invention (β-glucan content: 24%). The resulting fat spread wasevaluated for stability, smoothness and flavor. The results are shown inTable 1. The resulting fat spread underwent no separation of water whenstored for 1 month and had a smooth texture and a very good flavor.

COMPARATIVE EXAMPLE 7 Production of Comparative Fat Spread

A mixture consisting of 27.6 parts of hydrogenated fish oil. (meltingpoint: 36° C.), 18.4 parts of cotton seed oil, 52.3 parts of water, 1part of edible salt, 0.5 parts of defatted milk powder, 0.2 parts offlavor, and 0.3 parts of lecithin was emulsified and rapidly cooled forplasticization to prepare fat spread. The resulting fat spread wasevaluated for stability, smoothness and flavor for comparison. Theresults are shown in Table 2.

EXAMPLE 32 Production of Roux Type Curry Sauce Mix

Forty-four parts of wheat flour (cake flour) and 34 parts of theshortening obtained in Example 22 were pan-fried brown, and theresulting roux was mixed with 8 parts of a commercially available currypowder mix to obtain a curry sauce mix (β-glucan content: 0.95%)according to the present invention. The β-glucan was found uniformlydispersed.

EXAMPLE 33 Making of Cookies

Fifty parts of β-glucan-containing fat and oil composition-9 obtained inExample 9 and 50 parts of soft white sugar were kneaded into cream in ahobart mixer at a high speed for 6 minutes. A mixture of 15 parts net ofwhole eggs, 1 part of edible salt, and 0.5 parts of ammoniumhydrogencarbonate was added thereto, followed by mixing at a mediumspeed for 30 seconds. A hundred parts of sieved wheat flour was added,followed by mixing at a low speed for 30 seconds to prepare dough. Thedough was put into a cylinder of 6 cm in diameter, pressed out by 1 cm,and cut. The cut pieces of the dough were baked at 200° C. for 13minutes to obtain cookies (β-glucan content: 4.6%) according to thepresent invention. The β-glucan was found uniformly dispersed. Thecookies were evaluated for firmness and flavor. The results are shown inTable 1.

COMPARATIVE EXAMPLE 8 Making of Comparative Cookies

Fifty parts of a fat and oil mixture (30 parts of palm olein oil, 70parts of rapeseed oil, and 0.2 parts of protease-hydrolyzed egg yolk)and 50 parts of soft white sugar were kneaded into cream in a hobartmixer at a high speed for 6 minutes. To the cream was added a mixture of15 parts net of whole eggs, 1 part of edible salt, and 0.5 parts ofammonium hydrogencarbonate was added thereto, followed by mixing at amedium speed for 30 seconds. The resulting mixture was further processedin the same manner as in Example 33 to make cookies. The cookies wereevaluated for firmness and flavor. The results are shown in Table 2.

EXAMPLE 34 Making of Cookies

Fifty parts of β-glucan-containing fat and oil composition-14 obtainedin Example 14 and 40 parts of soft white sugar were kneaded into creamin a hobart mixer at a high speed for 6 minutes. Twenty parts of raisinpaste was added thereto, followed by mixing at a medium speed for 30seconds. Sieved millet powder was added, followed by mixing at a lowspeed for 30 seconds to prepare dough. The dough was put into a cylinderof 6 cm in diameter, pressed out by 1 cm, and cut. The cut pieces of thedough were baked at 160° C. for 15 minutes to obtain cookies (β-glucancontent: 2.5%) according to the present invention. The β-glucan wasfound uniformly dispersed. The cookies were evaluated for firmness andflavor. The results are shown in Table 1. While containing neither eggnor dairy products, the cookies had satisfactory texture.

COMPARATIVE EXAMPLE 9 Making of Comparative Cookies

A mixture of 20 parts of hydrogenated soybean oil (melting point: 45°C.), 35 parts of palm oil, 30 parts of cotton seed oil, and 0.2 parts ofsoybean lysolecithin was allowed to stand at 70° C. for 10 minuets,emulsified in a high-speed mixer, and rapidly cooled for plasticizationto prepare a fat and oil composition showing margarine-like physicalproperties. Fifty parts of the resulting fat and oil composition and 40parts of soft white sugar were kneaded into cream in a hobart mixer at ahigh speed for 6 minutes. Twenty parts of raisin paste was addedthereto, followed by mixing at a medium speed for 30 seconds. Theresulting mixture was further processed in the same manner as in Example34 to make cookies. The cookies were evaluated for firmness and flavor.The results of evaluation are shown in Table 2.

EXAMPLE 35 Production of Chocolate

Twelve parts of cacao mass, 45 parts of powdered sugar, 20 parts ofwhole milk powder, 13 out of 23 parts of cacao butter, and 2 parts ofsample G were put into a hobart mixer and mixed with a beater at amedium speed for 3 minutes. The mixture was rolled and conched toprepare a β-glucan-containing fat and oil composition (β-glucan content:1%) according to the present invention. As observed with the naked eye,the β-glucan was found uniformly dispersed in the composition. The restof cacao butter was added thereto and mixed to obtain chocolate mass.The chocolate mass was subjected to tempering, poured into a mold, andcooled down to obtain chocolate of the present invention, which wasevaluated for smoothness, firmness, and flavor. The results obtained areshown in Table 1. The resulting chocolate had good melt in the mouth anda good flavor.

COMPARATIVE EXAMPLE 10 Production of Comparative Chocolate

Twelve parts of cacao mass, 45 parts of powdered sugar, 20 parts ofwhole milk powder, 13 out of 23 parts of cacao butter, and 2 parts of afat and oil mixture (30 parts of palm olein oil, 70 parts of rapeseedoil, and 0.2 parts of protease-treated egg yolk) were put into a hobartmixer and mixed with a beater at a medium speed for 3 minutes. Themixture was rolled and conched to prepare a fat and oil composition. Therest of cacao butter was added thereto and mixed to obtain chocolatemass, which was further processed in the same manner as in Example 35 tomake chocolate. The resulting chocolate was evaluated for smoothness,firmness, and flavor. The results obtained are shown in Table 2.

EXAMPLE 36 Production of Chocolate

Twelve parts of cacao mass, 45 parts of powdered sugar, 20 parts ofwhole milk powder, 23 parts of cacao butter, and 20 parts ofβ-glucan-containing fat and oil composition-4 obtained in Example 4 wereput into a hobart mixer and mixed with a beater at a medium speed for 3minutes. The mixture was rolled and conched to prepare chocolate mass.The chocolate mass was subjected to tempering, poured into a mold, andcooled to obtain chocolate of the invention (β-glucan content: 15%). Theβ-glucan was found uniformly dispersed in the chocolate. The resultingchocolate was evaluated for smoothness, firmness, and flavor. Theresults obtained are shown in Table 1. The chocolate had good melt inthe mouth and a good flavor.

COMPARATIVE EXAMPLE 11 Production of Comparative Chocolate

Twelve parts of cacao mass, 45 parts of powdered sugar, 20 parts ofwhole milk powder, 23 parts of cacao butter, and 20 parts of palm oilwere put into a hobart mixer and processed in the same manner as inExample 36 to make chocolate. The resulting chocolate was evaluated forsmoothness, firmness, and flavor. The results obtained are shown inTable 2.

EXAMPLE 37 Production of Bread

Bread was made by using the β-glucan-containing margarine obtained inExample 26. A hundred parts of wheat flour, 3 parts of yeast, 4 parts ofsugar, 2 parts of edible salt, 6 parts of the margarine obtained inExample 26, and 60 parts of water were mixed in a hopper mixer at amixing temperature of 28° C. at a low speed for 2 minutes and then at ahigh speed for 4 minutes to prepare bread dough. The dough was allowedto ferment at 28° C. for 60 minutes and divided into 450 g portions,which were each formed into a ball and allowed to prove at 28° C. for 20minutes. The dough was passed through a sheeter three times, shaped, putinto a one-loaf pan, and finally proofed at 38° C. and 90% RH until itrose 2 cm above the lip of the pan. The final proofing took 42 minutes.The proofed dough was baked at 220° C. for 23 minutes to obtain a loafof bread (β-glucan content: 0.16%) according to the present invention.The β-glucan was found uniformly dispersed throughout the bread. Theresulting bread was evaluated for firmness and flavor. The resultsobtained are shown in Table 1. The resulting bread had softness, goodvolume and a satisfactory texture.

COMPARATIVE EXAMPLE 12 Production of Comparative Bread

Bread was made in the same manner as in Example 26, except for replacingthe margarine obtained in Example 26 with margarine prepared in the samemanner as in Example 26 but using no β-glucan (sample K). The resultingbread was evaluated for firmness and flavor. The results are shown inTable 2.

EXAMPLE 38 Production of Bread

A hundred parts of wheat flour, 3 parts of yeast, 4 parts of sugar, 2parts of edible salt, 2 parts of the β-glucan-containing fat and oilcomposition-19 obtained in Example 19, 4 parts of shortening, 30 partsof sample B, and 33 parts of water were mixed in a hopper mixer at amixing temperature of 28° C. at a low speed for 2 minutes and then at ahigh speed for 4 minutes to prepare bread dough. The dough was allowedto ferment at 28° C. for 60 minutes and divided into 450 g portions,which were each formed into a ball and allowed to prove at 28° C. for 20minutes. The dough was passed through a sheeter three times, shaped, putinto a one-loaf pan, and finally proofed at 38° C. and 90% RH until itrose 2 cm above the lip of the pan. The final proofing took 46 minutes.The proofed dough was baked at 210° C. for 30 minutes to obtain a loafof bread (β-glucan content: 2.5%) according to the present invention.The β-glucan was found uniformly dispersed throughout the bread. Theresulting bread was evaluated for firmness and flavor. The resultsobtained are shown in Table 1. The resulting bread had softness, goodvolume and a satisfactory texture.

COMPARATIVE EXAMPLE 13 Production of Comparative Bread

A hundred parts of wheat flour, 3 parts of yeast, 4 parts of sugar, 2parts of edible salt, 2 parts of a powdered fat and oil compositionprepared in the same manner as in Example 19 except for using noβ-glucan (sample G), 4 parts of shortening, and 63 parts of water weremixed in a hopper mixer at a mixing temperature of 28° C. at a low speedfor 2 minutes and then at a high speed for 4 minutes to prepare breaddough. The dough was further processed in the same manner as in Example38 to obtain a loaf of bread. The bread was evaluated for firmness andflavor. The results are shown in Table 2.

EXAMPLE 39 Production of Boiled Rice

Japonica rice cultivar Koshihikari produced in Niigata, Japan was washedwell with water. To 100 parts of washed rice were added 60 parts ofwater and 4 parts of the β-glucan-containing fat and oil composition-3obtained in Example 3. The rice was boiled in an electric rice cooker toobtain boiled rice of the present invention (β-glucan content: 0.51%).The β-glucan was found uniformly distributed. The boiled rice wasevaluated for firmness. The results are shown in Table 1. The resultingboiled rice had a light and soft texture.

COMPARATIVE EXAMPLE 14 Production of Comparative Boiled Rice

Japonica rice cultivar Koshihikari produced in Niigata, Japan was washedwell with water. To 100 parts of washed rice were added 60 parts ofwater and 4 parts of soybean oil. The rice was boiled in an electricrice cooker to obtain boiled rice, which was evaluated for firmness. Theresults are shown in Table 2.

EXAMPLE 40 Production of Popcorn

Into a pan were put 100 parts of popcorn kernels, 2 parts of ediblesalt, and 10 parts of the β-glucan-containing fat and oil composition-10obtained in Example 10, and the pan covered with a lid was heated onfire to make popcorn (β-glucan content: 0.38%) of the present invention.The β-glucan was found uniformly distributed. The resulting popcorn wasevaluated for smoothness. The results are shown in Table 1. The popcornhad a smooth and light texture.

EXAMPLE 41 Production of Tofu

Tofu was made by using the shortening prepared in Example 22. A hundredparts of soybeans having been soaked in water was ground together with140 parts of water, boiled at 100° C. for 5 minutes, transferred into acotton bag, and squeezed to obtain soy milk. To the soy milk were added3 parts of a coagulant (calcium sulfate) and 10 parts of the shorteningobtained in Example 22. The mixture was gently stirred, coagulated at75° C., and poured into a strainer lined with cotton cloth, left tostand for 30 minutes to obtain tofu (β-glucan content: 0.095%) accordingto the present invention. The β-glucan was found uniformly dispersed.The resulting tofu was evaluated for smoothness and flavor. The resultsare shown in Table 1. The resulting tofu had a good texture.

COMPARATIVE EXAMPLE 15 Production of Comparative Tofu

A hundred parts of soaked soybeans was ground together with 140 parts ofwater, boiled at 100° C. for 5 minutes, transferred into a cotton bag,and squeezed to obtain soy milk. To the soy milk were added 3 parts of acoagulant (calcium sulfate) and 10 parts of shortening prepared in thesame manner as in Example 22 except for using no β-glucan (sample G).The mixture was gently stirred, coagulated at 75° C., and poured into astrainer lined with cotton cloth, left to stand for 30 minutes to obtaintofu. The resulting tofu was evaluated for smoothness and flavor. Theresults are shown in Table 2.

EXAMPLE 42 Production of Soft Chocolate

A mixture consisting of 50 parts of sugar, 5 parts of cacao mass, 15parts of whole fat milk powder, 30 parts of the fat and oilcomposition-4 obtained in Example 4, 0.3 pails of lecithin, and 0.04parts of vanillin was subjected to rolling and conching in a usualmanner to obtain soft chocolate (β-glucan content: 13.5%) according tothe present invention. The β-glucan was found uniformly dispersed. Theresulting soft chocolate was evaluated for smoothness, firmness, andflavor. The results obtained are shown in Table 1. The resulting softchocolate underwent no blooming and had a good flavor.

COMPARATIVE EXAMPLE 16 Production of Comparative Soft Chocolate

A mixture consisting of 50 parts of sugar, 5 parts of cacao mass, 15parts of whole fat milk powder, 30 parts of palm oil, 0.3 parts oflecithin, and 0.04 parts of vanillin was subjected to rolling andconching in a usual manner to obtain soft chocolate, which was evaluatedfor smoothness, firmness, and flavor. The results obtained are shown inTable 2.

EXAMPLE 43 Production of Water-Free Cream

Thirty-five parts of the shortening prepared in Example 23, 45 parts ofsugar, 10 parts of a tasty powder, and 10 parts of milk powder weremixed to obtain water-free cream (β-glucan content: 1%) according to thepresent invention. The β-glucan was found uniformly dispersed. Theresulting water-free cream was evaluated for smoothness and flavor. Theresults are shown in Table 1. The water-free cream had good melt in themouth and a very good flavor.

COMPARATIVE EXAMPLE 17 Production of Comparative Water-Free Cream

Thirty-five parts of shortening prepared in the same manner as inExample 23 except for using no β-glucan (sample K), 45 parts of sugar,10 parts of a tasty powder, and 10 parts of milk powder were mixed toobtain water-free cream, which was evaluated for smoothness and flavor.The results are shown in Table 2.

EXAMPLE 44 Production of Whipped Cream for Cream Sandwiches

A mixture of 100 parts of the shortening prepared in Example 23 and 0.1parts of a monoglyceride was beaten into whipped cream having a specificgravity of 0.3. A hundred parts of sugar syrup was added, and the creamwas further beaten to prepare whipped cream having a specific gravity of0.65 (β-glucan content: 1.45%), which was for cream sandwiches. Theβ-glucan was found uniformly dispersed. The whipped cream was evaluatedfor smoothness and flavor. The results are shown in Table 1. Theresulting whipped cream had a very good flavor.

COMPARATIVE EXAMPLE 18 Production of Comparative Whipped Cream for CreamSandwiches

Whipped cream for cream sandwiches was made in the same manner as inExample 44, except for using 100 parts of shortening prepared in thesame manner as in Example 23 except for using no β-glucan (sample K).The resulting whipped cream was evaluated for smoothness and flavor. Theresults obtained are shown in Table 2.

EXAMPLE 45 Production of Hard Candy

Thirty-five parts of a fat and oil composition consisting of 100 partsof the β-glucan-containing fat and oil composition of Example 1, 100parts of the β-glucan-containing fat and oil composition-6 of Example 6,23 parts of a polyglycerol fatty acid ester, 14 parts of a glycerolfatty acid ester, and 4 parts of a sucrose fatty acid ester, 35 parts ofsugar, 8.5 parts of thick malt syrup, 1.5 parts of defatted milk powder,and 40 parts of water were mixed into an oil-in-water emulsion. Theemulsion was boiled down until the temperature reached 140° C. andfurther concentrated until the water content was reduced to 1.9%. Theresulting thick syrup was cooled and molded to obtain hard candyaccording to the present invention (β-glucan content: 12.5%). Theβ-glucan was found uniformly dispersed. The resulting hard candy wasevaluated for stability, smoothness, and flavor. The results are shownin Table 1. The resulting hard candy underwent no bleeding of oilycomponents during storage and had a good flavor.

COMPARATIVE EXAMPLE 19 Production of Comparative Hard Candy

Thirty-five parts of a fat and oil composition consisting of 100 partsof soybean oil, 100 parts of palm oil, 23 parts of a polyglycerol fattyacid ester, 14 parts of a glycerol fatty acid ester, and 4 parts of asucrose fatty acid ester, 35 parts of sugar, 8.5 parts of thick maltsyrup, 1.5 parts of defatted milk powder, and 40 parts of water weremixed into an oil-in-water emulsion. The emulsion was boiled down untilthe temperature reached 140° C. and further concentrated to watercontent of 1.9%. The resulting thick syrup was cooled and molded toobtain hard candy. The resulting hard candy was evaluated for stability,smoothness, and flavor. The results are shown in Table 2.

EXAMPLE 46 Production of Whipped Cream

In 50 parts of water heated to 60° C. were dissolved 5 parts of defattedmilk powder and 0.1 parts of sodium tripolyphosphate while stirring toprepare an aqueous phase. Separately, 10 parts of theβ-glucan-containing fat and oil composition-3 of Example 3, 20 parts ofthe β-glucan-containing fat and oil composition-4 of Example 4, and 15parts of the β-glucan-containing fat and oil composition-7 of Example 7were mixed to prepare an oil phase. The oil phase was mixed with theaqueous phase by stirring to prepare a preliminary emulsion. Thepreliminary emulsion was homogenized under a pressure of 5 MPa,sterilized in a VTIS sterilization apparatus at 142° C. for 4 seconds,re-homogenized under a pressure of 5 MPa, cooled to 5° C., and then agedin a refrigerator for 24 hours to give whipped cream (β-glucan content:13.6%) according to the present invention. The β-glucan was founduniformly dispersed. The resulting whipped cream was evaluated forstability, smoothness, and flavor. The results are shown in Table 1. Theresulting whipped cream was proved to have satisfactory qualities ofoverrun, emulsion stability, heat resistant shape retention, flavor,melt-in-the-mouth, and shapability in piping into rosettes.

COMPARATIVE EXAMPLE 20 Production of Comparative Whipped Cream

In 50 parts of water heated to 60° C. were dissolved 5 parts of defattedmilk powder and 0.1 parts of sodium tripolyphosphate while stirring toprepare an aqueous phase. Separately, 10 parts of soybean oil, 20 partsof palm oil, and 15 parts of rapeseed oil were mixed to prepare an oilphase. The oil phase was mixed with the aqueous phase by stirring toprepare a preliminary emulsion. The preliminary emulsion was furtherprocessed in the same manner as in Example 46 to obtain whipped cream,which was evaluated for stability and flavor. The results obtained areshown in Table 2.

EXAMPLE 47 Production of Milk Substitute Composition

In 64 parts of water heated to 60° C. were dissolved 25 parts ofdefatted milk powder, 0.2 parts of sodium hexametaphosphate, 0.2 partsof sodium citrate, and 0.3 parts of a sucrose fatty acid ester whilestirring to prepare an aqueous phase. To the aqueous phase were added 10parts of the β-glucan-containing fat and oil composition-17 of Example17 and 0.3 parts of a glycerol fatty acid ester and mixed by stirring toprepare a preliminary emulsion. The preliminary emulsion was homogenizedunder a pressure of 5 MPa, sterilized in a VTIS sterilization apparatusat 142° C. for 4 seconds, re-homogenized under a pressure of 15 MPa, andcooled to 5° C. to obtain a milk substitute composition (β-glucancontent: 2.2%) according to the present invention. The β-glucan wasfound uniformly dispersed. The milk substitute composition was evaluatedfor stability and flavor. The results are shown in Table 1. Theresulting milk substitute composition was proved satisfactory in bothflavor and emulsion stability.

COMPARATIVE EXAMPLE 21 Production of Comparative Milk SubstituteComposition

In 64 parts of water heated to 60° C. were dissolved 25 parts ofdefatted milk powder, 0.2 parts of sodium hexametaphosphate, 0.2 partsof sodium citrate, and 0.3 parts of a sucrose fatty acid ester whilestirring to prepare an aqueous phase. To the aqueous phase were added 10parts of a fat and oil composition prepared in the same manner as inExample 17 except for adding no β-glucan (sample H) and 0.3 parts of aglycerol fatty acid ester and mixed by stirring to prepare a preliminaryemulsion. The preliminary emulsion was further processed in the samemanner as in Example 47 to give a milk substitute composition. Theresulting milk substitute composition was evaluated for stability andflavor. The results are shown in Table 2.

EXAMPLE 48 Production of Food (Margarine) with Prophylactic Effects onHabitual Diseases

Ten parts of hydrogenated soybean oil (melting point: 45° C.), 35 partsof palm oil, 10 parts of the β-glucan-containing fat and oilcomposition-3 obtained in example 3, 30 parts of an interesterified oilcontaining at least 10% of plant sterol or plant sterol fatty acidesters, 13.3 parts of sample K, 1 part of edible salt, 0.5 parts ofdefatted milk powder, and 0.2 parts of flavor were emulsified andrapidly cooled for plasticization to make margarine having a cholesterollowering effect (β-glucan content: 10%) according to the presentinvention. The β-glucan was found uniformly dispersed. The margarinehaving a cholesterol lowering effect was evaluated for smoothness andflavor. The results are shown in Table 1. The resulting margarine hadgood melt in the mouth and a good flavor.

COMPARATIVE EXAMPLE 22 Production of Comparative Food (Margarine) withProphylactic Effects on Habitual Diseases

Ten parts of hydrogenated soybean oil (melting point: 45° C.), 35 partsof palm oil, 10 parts of soybean oil, 30 parts of an interesterified oilcontaining at least 10% of plant sterol or plant sterol fatty acidesters, 13.3 parts of water, 1 part of edible salt, 0.5 parts ofdefatted milk powder, and 0.2 parts of flavor were emulsified andrapidly cooled for plasticization to make margarine having a cholesterollowering effect, which was evaluated for smoothness and flavor. Theresults are shown in Table 2.

EXAMPLE 49 Production of Drug with Prophylactic Effects on HabitualDiseases

Three parts of high purity DHA (purity: 98%; POV: 1.0 meq/kg or less)containing 4000 ppm of α-tocopherol, 20 parts of sample K, and 10 partsof casein sodium were emulsified in a high-speed mixer in a nitrogenatmosphere and spray dried to prepare a powdered drug with prophylacticeffects on habitual diseases (β-glucan content: 36.4%) according to thepresent invention. The β-glucan was found uniformly dispersed. Theresulting drug with prophylactic effects on habitual diseases wasevaluated for stability. The results are shown in Table 1. The POV ofthe drug was 0.8 meq/kg, proving the drug to be excellent inantioxidation stability.

COMPARATIVE EXAMPLE 23 Production of Comparative Drug with ProphylacticEffects on Habitual Diseases

Three parts of high purity DHA (purity: 98%; POV: 1.0 meq/kg or less)containing 4000 ppm of α-tocopherol, 20 parts of water, and 10 parts ofcasein sodium were emulsified in a high-speed mixer in a nitrogenatmosphere and spray dried to prepare a powdered drug with prophylacticeffects on habitual diseases, which was evaluated for stability. Theresults are shown in Table 2. The POV of the resulting drug was 1.4meq/kg, indicating inferior antioxidation stability.

In Examples and Comparative Examples described supra, stability andtexture (smoothness, firmness, and flavor) of products were evaluated asfollows. The mark “−” in Tables 1 and 2 indicates no evaluation made.

How to Evaluate the Stability

Stability was evaluated by visually inspecting for any change inappearance after 1 month storage at 5° C. and rated A to C according tothe following standards.

<Evaluation Standards>

-   A: Excellent in stability.-   B: Change in appearance, such as slight phase separation, observed.-   C: Phase separation observed.

How to Evaluate the Texture

The texture was organoleptically evaluated by 10 panel members and ratedA to C according to the following standards. The rating given to asample by the greatest number out of 10 panel members was the rating ofthe sample.

<Evaluation Standards>

-   1) Smoothness-   A: Very smooth-   B: Smooth-   C: Not smooth-   2) Firmness-   A: Very soft-   B: Soft-   C: Not soft-   3) Flavor-   A: Superior-   B: Slightly inferior

C: Inferior TABLE 1 Texture Example Stability Smoothness Firmness Flavor22 — A — A 23 — A — A 24 A A — A 25 A A — A 26 A A — A 27 A — — A 28 A —— A 29 A A — A 30 A A — A 31 A A — A 33 — — A A 34 — — A A 35 — A A A 36— A A A 37 — — A A 38 — — A A 39 — — A — 40 — A — — 41 — A — A 42 — A AA 43 — A — A 44 — A — A 45 A A — A 46 A A — A 47 A — — A 48 — A — A 49 A— — —

TABLE 2 Compara. Texture Example Stability Smoothness Firmness Flavor 1— B — C 2 A A — B 3 B — — B 4 B — — A 5 C A — B 6 C B — B 7 B A — B 8 —— B B 9 — — B B 10 — A B B 11 — A B B 12 — — B A 13 — — B A 14 — — B —15 — A — B 16 — A B C 17 — A — C 18 — A — C 19 B A — B 20 B — — C 21 B —— B 22 — B — C 23 C — — —

The novel microorganisms according to the present invention will then bedescribed more specifically with reference to Examples. Test Examples 1and 2 show screening for obtaining strains of the microorganisms. TestExamples 3 to 7 show the mycological properties of the strain ADK-34.Examples 50 to 52 demonstrate production of β-glucan using ADK-34.Analyses were conducted in the same manner as described supra inAnalysis Examples 1 to 3.

TEST EXAMPLE 1 Strain Screening Method I

Microorganisms attaching to and growing on the surface of a broad rangeof commercially available foods that are usually eaten without cookingand mainly including Japanese traditional preserved foods were separatedand screened to find β-glucan-producing strains. The screening method isdescribed below.

A food (subject sample) was put in a sterilized petri dish, and 10 ml ofsterilized PBS was added. The surface of the sample was repeatedlywashed with the added PBS using a sterilized dropping pipette. Theresulting washing was 10 to 100-fold diluted with sterilized PBS. A 200μL portion of each dilution was put on an agar plate, spread with aspreader, and incubated at room temperature for two weeks. The agarplate was prepared by solidifying 20 ml of YM medium (Difco) containing100 μg/ml of chloramphenicol and 1.5 wt % agar. Out of about 20,000grown colonies those having the following properties were picked fromthe plate: those which were creamy white in the beginning of growth andgradually became glossy as a whole and wet; those which were wet andglossy as a whole with a slightly yellow to brown center or an yellow tobrown edge; those assuming creamy white to pink as a whole; and thosewhich gradually became greenish black and fuzzy. Colonies that were pinkas a whole, non-glossy, and convex with non-spreading edge wereexcluded. The picked colonies were again incubated for 7 days for singlestrain isolation. Colonies that were found to have formed blastoconidiaand/or shown yeast-like growth under microscopic observation wereselected, and those observed to have conidiophores were screened out(results of first screening).

The strains obtained by the first screening were liquid-cultured. Thatis, each strain was cultured in YM medium containing 5 wt % sucrose at26° for 4 days using a 24-well microplate. Those cultures which wereviscous and in which the strain was uniformly dispersed were left(results of second screening).

A single strain isolated from the results of the second screening wasinoculated into YM liquid medium and cultured at 26° C. for 96 hours. Tothe culture was added an equivalent amount of distilled water, and theculture was sterilized at 121° C. for 20 minutes and centrifuged at8,000 rpm to obtain the supernatant liquid. To 30 μl of the liquid wasadded twice its volume of ethanol, followed by centrifugation at 1000rpm for 10 minutes to separate into the precipitate and the medium. Tothe precipitate was added 100 μl of distilled water, and the totalpolysaccharide content was measured by the phenol-sulfuric acid method.The total polysaccharide content in the medium was also measured in thesame manner. The cultures whose total polysaccharide content was higherin the precipitate than in the medium were judged positive. The strainsjudged positive are the results of third screening. I The firstscreening of approximately 20,000 colonies grown from strains separatedfrom subject samples gave 180 strains. The second screening resulted inisolation of 50 strains. The third screening provided 14 positives. Thestrains obtained by the third screening and three other strainspurchased from Institute for Fermentation, Osaka, i.e., IFO-4466,IFO-6353, and IFO-7757 were each cultured to obtain their culturesolutions. The β-glucan content of each culture solution was measured bythe method taught in Analysis Example 1. The purity with respect toproduced polysaccharide pullulan in the culture solution was alsomeasured by the method shown in Analysis Example 3. The results obtainedare shown in Table 3 below. The polysaccharide produced by ADK-34 wasnot digested by pullulanase, which indicates that the amount of thepolysaccharide approximately agrees with the β-glucan content asdetermined. Namely, it was concluded that this strain produces β-glucanwith high purity. TABLE 3 Absorbance (490 nm) β-Glucan Before EnzymeAfter Enzyme Content Strain Treatment Treatment Purity (%) (mg/ml) ADK-11.067 0.389 36.5 0.12 ADK-4 1.183 0.504 42.6 0.23 ADK-5 1.655 1.042 631.34 ADK-6 1.364 0.764 56 0.649 ADK-8 1.344 0.914 68 0.674 ADK-10 1.4690.646 44 0.357 ADK-17 1.224 0.759 62 0.411 ADK-20 1.003 0.552 55 0.264ADK-24 1.141 0.776 68 0.888 ADK-27 1.225 0.882 72 0.954 ADK-28 1.3770.565 41 0.5 ADK-31 1.419 0.426 30 0.311 ADK-34 1.366 1.369 100 1.89ADK-42 1.154 0.427 37 0.44 IFO-6353 1.455 0.757 52 0.743 IFO-7757 0.9750.722 74 0.684 IFO-4466 0.736 0.521 71 0.701 Pullulan 0.755 0.016 3.9 01 mg/ml Pullulan 0.411 0.009 2.2 0 0.5 mg/ml

TEST EXAMPLE 2 Strain Screening Method II

Strain screening was carried out in the same manner as in Test Example1, except for using media containing 10 μg/ml of the antibioticcycloheximide. As a result, the first screening of 4,000 colonies grownfrom strains separated from subject samples gave 30 strains. The secondscreening resulted in isolation of 10 strains. The third screeningprovided 3 positive strains (ADK-71, ADK-77, and ADK-82). The strainsobtained by the third screening were tested in the same manner as inTest Example 1 to determine the β-glucan content and the purity withrespect to the produced polysaccharide pullulan. The results obtainedare shown in Table 4.

Furthermore, the three strains obtained by the third screening wereidentified through morphological characterization. The strains were eachinoculated into YM agar medium and cultivated at 26° C. for 7 days. Allthe strains formed wholly glossy with a yellow pigmentation in thecentral portion and wet colonies. The plates were then stored at 4° C.for 7 days, whereupon the central yellow changed to blackish green. Thecolonies of ADK-71 and ADK-77 did not change in the area other than thecentral portion and remained white to pink. The colonies of ADK-82turned black as a whole. Separately, when the three strains were eachcultivated in YM liquid medium at 26° C. for 3 days, every strain wasfound to have formed blastoconidia and shown yeast-like growth undermicroscopic observation. In addition, the fungi of each of the threestrains cultivated in YM agar medium at 26° C. for 7 days were observedto have developed chains of conidia but with no conidiophores. Fromthese observations, the three strains were identified to beAureobasidium pullulans species. TABLE 4 Absorbance (490 nm) β-GlucanBefore Enzyme After Enzyme Content Strain Treatment Treatment Purity (%)(mg/ml) ADK-71 0.882 0.893 101 1.22 ADK-77 0.741 0.842 114 0.936 ADK-820.633 0.655 103 0.854 Pullulan 0.768 0.015 2 0 1 mg/ml Pullulan 0.3930.005 1.3 0 0.5 mg/ml

TEST EXAMPLE 3 Morphological and Cultural Properties

ADK-34, chosen from the strains obtained in Test Example 1, was culturedin YM medium (1 wt % glucose, 0.5 wt % peptone, 0.3 wt % yeast extract,0.3 wt % malt extract; pH 6.0) at 26° C. for 3 days. Microscopicobservation revealed the following. The cell size was 2 to 2.5 μm inwidth and 5 to 10 μm in length. The cells were colorless, oval, andsmooth on the surface. No motility was observed. The hyphae were sparse,non-uniform, colorless, and smooth on the surface. The width of thehyphae was 2.5 μm. Formation of blastoconidia which was similar to yeastbudding was observed.

TEST EXAMPLE 4 Agar Plate Cultivation

ADK-34, one of the strains obtained in Test Example 1, was cultured on apotato-dextrose-agar medium (Eiken) at 26° C. for 7 days. At day 3, thestrain showed good growth, and the colonies were circular with a roughedge, glossy as a whole, smooth, and white. At day 5, the colonies wereslightly grayish white with a smooth surface and showed yeast-likegrowth. At day 7, the surface of colonies turned pink. After 7 daycultivation at 26° C., the plate was refrigerated at 4° C. for 7 days,whereupon the pink pigmentation became slightly deeper but with nochange of the individual colonies as a whole.

TEST EXAMPLE 5 Liquid Cultivation

ADK-34, one of the strains obtained in Test Example 1, was cultured inYM medium to examine the optimum growth temperature and pH. The optimumgrowth temperature was 26° C. The optimum growth pH was 5.0 to 7.0, thepH at the beginning of growth was 6.2, and the pH after the end ofculturing was 7.5. A preferred growth temperature range was 20° to 30°C. The optimum growth temperature was 26° C. The permissible growthtemperature range was 5° to 40° C. The strain decomposed hexoses such asglucose, fructose, and mannose, disaccharides such as sucrose, andstarch. With any of these carbon sources the culture became viscous andhad a specific aroma.

As a result of Test Examples 3 to 5, the ADK-34 strain, one of themicroorganisms according to the present invention, was identified to bea strain belonging to the genus Aureobasidium from its mycologicalcharacteristics.

TEST EXAMPLE 6 Cycloheximide Resistance Test

The strains obtained by the screening in Test Examples 1 and 2 andIFO-4466, IFO-6353, and IFO-7757 purchased from Institute forFermentation, Osaka, i.e., were tested for cycloheximide resistance asfollows. Each strain was grown on YM agar medium (Difco) at 26° C. for 5days. YM agar media (Difco) containing 5, 10, 20, 40, and 80 μg/ml ofcycloheximide were prepared. The grown strain was inoculated into eachof the cycloheximide-containing media using a sterile toothpick andincubated at 26° C. for 10 days. The plates were examined for colonies.The diameters of the colonies, if formed, were measured. The results areshown in Table 5. TABLE 5 (unit: Cycloheximide Concentration (μg/ml)mm)Strain 0 5 10 20 40 80 ADK-1 16.5 0 0 0 0 0 ADK-4 17.3 8.5 0 0 0 0ADK-5 15.6 6.3 0 0 0 0 ADK-6 14.4 7.1 0 0 0 0 ADK-8 16.8 7.7 1.1 0 0 0ADK-10 14.9 8.5 0 0 0 0 ADK-17 12.7 6.7 0 0 0 0 ADK-20 13.3 6.2 0 0 0 0ADK-24 15.6 9.1 2.5 0 0 0 ADK-27 17.4 10.3 0 0 0 0 ADK-28 15.4 7.1 0 0 00 ADK-31 16.8 6.3 1.7 0 0 0 ADK-34 15.5 13.6 6.7 5.1 3.7 0.5 ADK-42 12.72.4 0 0 0 0 ADK-71 13.7 7.6 4.1 1.8 1.2 0 ADK-77 15.4 9.5 4.1 0.8 0 0ADK-82 16.8 10.4 2.9 1.5 1.2 0 IFO-7757 17.5 0 0 0 0 0 IFO-6353 15.6 7.32.3 0 0 0 IFO-4466 14.8 4.4 0 0 0 0

As is apparent from Tables 3 and 5, IFO-4466, IFO-6353, and IFO-7757 donot have resistance to cycloheximide, and the β-glucan produced by themhave low purity with respect to pullulan. It is obvious from Tables 3 to5, on the other hand, that those isolates isolated front foods which areresistant to cycloheximide, i.e., ADK-34, ADK-71, ADK-77, and ADK-82produce β-glucan with good purity with respect to pullulan and that manyof those isolates which are not resistant to cycloheximide produceβ-glucan but at low purity with respect to pullulan.

TEST EXAMPLE 7 Gene Analysis on 18S rRNA Gene

The sequence of 1732 base pairs of the 18S rRNA gene of ADK-34, obtainedby the screening, was determined as follows. ADK-34 was shake-culturedin a potato-dextrose agar (Difco), and the culture was centrifuged. Thesolid was washed three times with distilled water to obtain fungi forDNA extraction. The fungi were cellularly disrupted using FastPrep FP120(from Q-Biogene) and FastDNA-kit (from Q-Biogene), and genomic DNA wasisolated using Dneasy Plant Mini Kit (from Qiagen). PCR amplificationwas carried out using the genomic DNA as a template, primers NS1 andNS8, and Ready-To-Go PCR Beads (Amersharm-Pharmasia Biotech).

The sequence (5′→3′) of the primers NS1 and NS8 was GTAGTCATATGC=TGTCTC(NS1) and TCCGCAGGTTCACCTACGGA (NS8), respectively. As a thermal cyclergeneAmp PCR System 9600 (Applied Biosystems) was used. After completionof the PCR reaction, the PCR product was purified using QlAquick PCRPurification Kit (Qiagen). The resulting DNA fragment was subjected todirect sequencing reaction, and the base sequence was analyzed on ABIPrism 377 DNA Sequencer (Applied Biosystems). A DNA data base (DNA DataBank of Japan, DDBJ) was searched for homologous sequences using theBLAST program.

The results of sequence analysis are shown in Sequence Listing, SEQ IDNo. 1. The data base was searched based on the determined base sequenceto examine homology between ADK-34 and homologous strains. The searchresults are shown in Tables 6 through 8. The determined base sequence(SEQ ID No. 1) and the results of the homology search (Tables 6 to 8)revealed 100% homology (perfect agreement) of ADK-34 with Aureobasidiumpullulans. The present strain was thus identified to be Aureobasidiumpullulans. TABLE 6 Sequences producing significant alignments: (bits)Value AY030322|AY030322.1 Aureobasidium pullulans 18S ribosomal RNA g .. . 3433 0.0 1732/1732 (100%) M55639|M55639.1 Aureobasidium pullulans16S-like ribosomal RNA . . . 3429 0.0 1731/1732 (99%) U42474|U42474.1Dothidea insculpta 18S small subunit ribosomal . . . 3237 0.0 1699/1721(98%) U42475|U42475.1 Dothidea hippophaeos 18S small subunit ribosoma .. . 3221 0.0 1691/1713 (98%) U77668|U77668.1 Coccodinium bartschii 18Sribosomal RNA gene, p . . . 3178 0.0 1690/1719 (98%) AF258607|AF258607.1Scytalidium hyalinum strain IP252699 18S ri . . . 3158 0.0 1699/1733(98%), AF258606|AF258606.1 Scytalidium hyalinum strain IP151783 18S ri .. . 3158 0.0 1699/1733 (98%), AB041250|AB041250.1 Phyllosticta pyrolaegene for 18S rRNA, par . . . 3158 0.0 1699/1733 (98%),AB041249|AB041249.1 Guignardia endophyllicola gene for 18S rRNA . . .3150 0.0 1698/1733 (97%), AB041248|AB041248.1 Guignardia endophyllicolagene for 18S rRNA . . . 3150 0.0 1698/1733 (97%), AB041247|AB041247.1Guignardia endophyllicola gene for 18S rRNA . . . 3150 0.0 1698/1733(97%) Y11716|Y11716.1 P. dematioides 18S rRNA gene. 3148 0.0 1697/1732(97%), U42477|U42477.1 Botryosphaeria ribis 18S small subunit ribosoma .. . 3144 0.0 1689/1722 (98%), Y18702|Y18702.1 Sarcinomyces petricola 18SrRNA gene, strain CB . . . 3108 0.0 1693/1733 (97%), D49656|D49656.1Lasioderma serricorne yeast-like symbiote DNA f . . . 3102 0.0 1692/1733(97%), AJ224362|AJ224362.1 Bulgaria inquinans 18S rDNA. 3094 0.01691/1733 (97%), AF088239|AF088239.1 Lecidea fuscoatra 18S ribosomalRNA, partia . . . 3033 0.0 1666/1710 (97%), Y18693|Y18693.1 Hortaeawerneckii 18S rRNA gene, strain CBS 107 . . . 3027 0.0 1679/1731 (96%)AF088253|AF088253.1 Umbilicaria subglabra 18S ribosomal RNA, pa . . .3015 0.0 1648/1689 (97%), Y11355|Y11355.1 S. crustaceus 18S rRNA gene.3001 0.0 1674/1731 (96%), U42478|U42478.1 Sporormia lignicola 18S smallsubunit ribosomal . . . 2991 0.0 1665/1717 (96%) AF184755|AF184755.1Metus conglomeratus small subunit ribosomal . . . 2991 0.0 1678/1733(96%), AB016175|AB016175.1 Euascomycetes sp. K89 gene for 18S rRNA, pa .. . 2958 0.0 1680/1733 (96%), AF184753|AF184753.1 Cladonia rangiferinasmall subunit ribosma . . . 2976 0.0 1676/1733 (96%),AF140236|AF140236.1 Stereocaulon paschale small subunit ribosom . . .2976 0.0 1676/1733 (96%), AB015778|AB015778.1 Pseudogymnoascus roseus18S rRNA gene, part . . . 2972 0.0 1640/1687 (97%) AF184756|AF184756.1Pilophorus cereolus small subunit ribosomal . . . 2968 0.0 1675/1733(96%), AF184761|AF184761.1 Stereocaulon vesuvianum small subunit ribos .. . 2960 0.0 1674/1733 (96%), AF184754|AF184754.1 Heterodea muellerismall subunit ribosomal . . . 2960 0.0 1674/1733 (96%),AF168167|AF168167.1 Dark septate endophyte DS16b 18S ribosomal . . .2960 0.0 1661/1713 (96%), AF184757|AF184757.1 Pilophorus robustus smallsubunit ribosomal . . . 2952 0.0 1673/1733 (96%), AF117984|AF117984.1Hypogymnia physodes nuclear small subunit r . . . 2952 0.0 1665/1720(96%), AF088246|AF088246.1 Rhizocarpon geographicum 18S ribosomal RNA .. . 2946 0.0 1643/1693 (97%), AF241544|AF241544.1 Cladonia sulphurinasmall subunit ribosomal . . . 2944 0.0 1672/1733 (96%),AB015776|AB015776.1 Byssoascos striatosporus 18S rRNA gene, par . . .2940 0.0 1636/1687 (96%)

TABLE 7 Sequences producing significant alignments: (bits) ValueAF140233|AF140233.1 Alectoria sarmentosa small subunit ribosoma . . .2938 0.0 1671/1733 (96%), U70960|U70960.1 Pilophorus acicularis 18Ssmall subunit ribosom . . . 2936 0.0 1671/1733 (96%),AF085471|AF085471.1 Baeomyces rufus 18S small subunit ribosomal . . .2932 0.0 1638/1691 (96%) U70961|U70961.1 Stereocaulon ramulosum 18Ssmall subunit riboso . . . 2928 0.0 1670/1733 (96%), AF088238|AF088238.1Lasallia rossica 18S ribosomal RNA, partial . . . 2926 0.0 1654/1708(96%), Y14210|Y14210.1 Monilinia laxa 18S rRNA gene, exon 1. partial.2916 0.0 1636/1691 (96%) U42476|U42476.1 Botryosphaeria rhodina 18Ssmall subunit riboso . . . 2914 0.0 1619/1671 (96%), U86692|U86692.1Stenocybe pullatula 18S SSU ribosomal RNA, part . . . 2910 0.0 1640/1696(96%), AF117992|AF117992.1 Xanthoparmelia conspersa nuclear small subu .. . 2910 0.0 1664/1728 (96%), AF085475|AF085475.1 Cladoniasubcervicornis 18S small subunit r . . . 2910 0.0 1637/1692 (96%),AF085465|AF085465.1 Stereocaulon taeniarum 18S small subunit ri . . .2910 0.0 1634/1688 (96%), AB015787|AB015787.1 Oidiodendron tenuissimum18S rRNA gene, iso . . . 2910 0.0 1634/1688 (96%), AF140235|AF140235.1Cornicularia normoerica small subunit ribos . . . 2908 0.0 1663/1727(96%), AB015777|AB015777.1 Myxotrichum deflexum 18S rRNA gene, isolate .. . 2908 0.0 1632/1687 (96%) AF184759|AF184759.1 Psora decipiens smallsubunit ribosomal RNA . . . 2904 0.0 1667/1733 (96%),AF117981|AF117981.1 Neophyllis melacarpa nuclear small subunit . . .2904 0.0 1667/1733 (96%), AF088251|AF088251.1 Stereocaulon ramulosum 18Sribosomal RNA, p . . . 2904 0.0 1652/1713 (96%), AF088245|AF088245.1Pseudevernia cladoniae 18S ribosomal RNA, p . . . 2902 0.0 1638/1696(96%) AF201452|AF201452.1 Rhytidhysteron rufulum 18S ribosomal RNA ge .. . 2900 0.0 1578/1615 (97%), AF274110|AF274110.1 Lepolichen coccophons18S ribosomal RNA ge . . . 2898 0.0 1660/1725 (96%), AF117991|AF117991.1Pleurosricta acetabulum nuclear small subun . . . 2898 0.0 1664/1730(96%), U43463|U43463.1 Mycosphaerella mycopappi small subunit nuclear .. . 2894 0.0 1664/1732 (96%) AF085466|AF085466.1 Stereocaulon vesuvianum18S small subunit r . . . 2894 0.0 1632/1688 (96%), AB033475|AB033475.1Blumeria graminis f. sp. bromi gene for 18S . . . 2894 0.0 1660/1724(96%), U42485|U42485.1 Lophiostoma crenatum 18S small subunit ribosoma .. . 2892 0.0 1655/1719 (96%), AF053726|AF053726.1 Kirschsteiniotheliamaritima small subunit . . . 2890 0.0 1659/1726 (96%)AF201455|AF201455.1 Tubeufia helicoma 18S ribosomal RNA gene, p . . .2886 0.0 1586/1628 (97%), AF241541|AF241541.1 Xanthoria parietina smallsubunit ribosomal . . . 2884 0.0 1653/1719 (96%) U70959|U70959.1Leifidium tenerum 18S small subunit ribosomal R . . . 2880 0.0 1664/1733(96%), AF282910|AF282910.1 Lichinella cribellifera 18S small subunit r .. . 2880 0.0 1667/1733 (96%), AF117986|AF117986.1 Cetraria islandicanuclear small subunit ri . . . 2880 0.0 1646/1709 (96%), L37540|L37540.1Porpidia crustulata (Ach.) Hertel and Knoph nuc . . . 2878 0.0 1570/1608(97%), AF184751|AF184751.1 Cladia retipora small subunit ribosomal RNA .. . 2874 0.0 1636/1697 (96%),

TABLE 8 Sequences producing significant alignments: (bits) ValueAF282913|AF282913.1 Peltula obscurans 18S small subunit ribosom . . .2872 0.0 1663/1732 (96%), AF085474|AF085474.1 Pycnothelia papillaria 18Ssmall subunit ri . . . 2872 0.0 1618/1673 (96%), AB033479|AB033479.1Leveillula taurica gene for 18S ribosomal RNA . . . 2855 0.0 1654/1720(96%), AF117985|AF117985.1 Parmelia saxatilis nuclear small subunit ri .. . 2859 0.0 1653/1722 (95%), AF088254|AF088254.1 Xanthoria elegans 18Sribosomal RNA, partia . . . 2865 0.0 1655/1724 (95%),AF117988|AF117988.1 Usnea florida nuclear small subunit ribosom . . .2853 0.0 1635/1699 (96%), U42483|U42483.1 Herpotrichia juniperi 18Ssmall subunit ribosom . . . 2847 0.0 1649/1720 (95%) AF140234|AF140234.1Alectoria ochroleuca small subunit ribosoma . . . 2847 0.0 1598/1652(96%) AF091587|AF091587.1 Scoliciosporum umbrimum 18S ribosomal RNA, g .. . 2847 0.0 1625/1688 (96%) AF085469|AF085469.1 Pilophorus acicularis18S small subunit rib . . . 2845 0.0 1613/1671 (96%),AF010590|AF010590.1 Ascozonus woolhopensis SSU ribosomal RNA, ge . . .2841 0.0 1654/1727 (95%), AF117990|AF117990.1 Vulpicida juniperinanuclear small subunit . . . 2831 0.0 1624/1688 (96%), Z30239|Z30239.1 S.flavida gene for 18S ribosomal RNA. 2819 0.0 1638/1711 (95%),AB016174|AB016174.1 Geomyces pannorum gene for 18S rRNA, partia . . .2809 0.0 1544/1588 (97%), AB016173|AB016173.1 Geomyces asperulatus genefor 18S rRNA, par . . . 2809 0.0 1544/1588 (97%), AF117987|AF117987.1Evernia prunastri nuclear small subunit rib . . . 2807 0.0 1621/1688(96%), AF184749|AF184749.1 Bunodophoron australe small subunit ribosom .. . 2805 0.0 1619/1687 (95%) AF241540|AF241540.1 Caloplacaflavorubescens small subunit ribo . . . 2795 0.0 1624/1694 (95%),AF091583|AF091583.1 Lecidella meiococca 18S ribosomal RNA gene, . . .2795 0.0 1626/1697 (95%), AF282914|AF282914.1 Pterygiopsis guyanensis18S small subunit r . . . 2771 0.0 1654/1734 (95%), U72713|U72713.1Cladia aggregate 18S small subunit ribosomal RN . . . 2755 0.0 1597/1665(95%), AF091589|AF091589.1 Lecania cyrtella 18S ribosomal RNA gene, pa .. . 2753 0.0 1635/1713 (95%), AF085468|AF085468.1 Allocetrariamadreporiformis 18S small subu . . . 2732 0.0 1536/1592 (96%)AF088250|AF088250.1 Squamarina lentigera 18S ribosomal RNA, par . . .2692 0.0 1623/1710 (94%), AF201453|AF201453.1 Aliquandostipitekhaoyaiensis 18S ribosomal . . . 2684 0.0 1603/1684 (95%),AF258605|AF258605.1 Scytalidium dimidiatum strain IP252899 18S . . .2615 0.0 1374/1391 (98%), AF258604|AF258604.1 Scytalidium dimidiatumstrain IP252799 18S . . . 2615 0.0 1374/1391 (98%), AF258603|AF258603.1Scytalidium dimidiatum strain IP127881 18S . . . 2615 0.0 1374/1391(98%), AB033477|AB033477.1 Arthrocladiella mougeotii gene for 18S ribo .. . 2613 0.0 1418/1450 (97%), U45438|U45438.1 Amylocarpus encephaloidessmall subunit rRNA gene . . . 2605 0.0 1441/1481 (97%),AF274113|AF274113.1 Coccotrema pocillarium 18S ribosomal RNA ge . . .2375 0.0 1351/1401 (96%), AF184752|AF184752.1 Cladonia gracilis subsp.turbinate small su_. . . 2226 0.0 1239/1277 (97%),

TEST EXAMPLE 8 ITS-5.8S rRNA Gene

The sequence of 563 or 564 base pairs of the ITS-5.8S rRNA gene ofADK-34 obtained by the screening, IFO-6353, and IFO-7757 was determinedas follows. Each strain was shake-cultured in a potato-dextrose agar(Difco), and the culture was centrifuged and washed three times withdistilled water to obtain fungi for DNA extraction. The fungi werecellularly disrupted using FastPrep FP120 (from Q-Biogene) andFastDNA-kit (from Q-Biogene), and genomic DNA was isolated using DneasyPlant Mini Kit (from Qiagen). PCR amplification was carried out usingthe genomic DNA as a template, primers ITS5 and ITS4, and Realdy-To-GoPCR Beads (Amersharm-Pharmasia Biotech).

The sequence (5′→3′) of the primers ITS4 and ITS 5 wasTCCTCCGCITATTGATATGC (ITS4) and GGAAGTAAAAGTCGTAACAAGG (ITS5),respectively. As a thermal cycler geneAmp PCR System 9600 (AppliedBiosystems) was used. After completion of the PCR reaction, the PCRproduct was purified using QIAquick PCR Purification Kit (Qiagen). Theresulting DNA fragment was subjected to direct sequencing reaction, andthe base sequence was analyzed on ABI Prism 377 DNA Sequencer (AppliedBiosystems). A DNA data base (DNA Data Bank of Japan, DDBJ) was searchedfor homologous sequences using the BLAST program.

The results of ADK-34, IFO-6353, and IFO-7757 are shown in SequenceListing, SEQ ID Nos. 2, 3, and 4, respectively. The data base wassearched based on the determined base sequences to examine homologybetween these strains and their homologous strains. The search resultsare shown in Tables 9 and 10. On comparing the determined basesequences, ADK-34 was found different from either of IFO-6353 andIFO-7757 (not in complete agreement). The homology (similarity) betweenADK-34 and IFO-6353 was 98%, and that between ADK-34 and IFO-7757 was98%. The homology search results (Tables 9 and 10) indicated that (1)there is no report on a strain whose 563 bp in the ITS-5.8S regioncompletely agree with those of ADK-34; (2) IFO-6353 showed 100% homology(perfect agreement) with the reported Aureobasidium pullulans strain,Accession No. AJ276062 (see Table 11); (3) IFO-7757 showed 99% homologywith the reported Aureobasidium pullulans strain, Accession No. AJ276062(see Table 12); and (4) the homology between ADK-34 and Accession No.AJ276062 was 98%.

From these results, ADK-34 was judged to be a new strain, differing fromthe reported Aureobasidium pullulans strains in part of the basesequence in the ITS-5.8S region. TABLE 9 Sequences producing significantalignments: (bits) Value AF013229|AF013229.1 Aureobasidium pullulans 18Sribosomal RNA g . . . 997 0.0 555/567 (97%), AY029406|AY029406.1 Astasialonga internal transcribed spacer 1 . . . 989 0.0 538/546 (98%),AJ276062|AJ276062.1 Aureobasidium pullulans 5.8S rRNA gene and . . . 9710.0 517/526 (98%) AF121287|AF121287.1 Aureobasidium pullulans var.melanigenum st . . . 971 0.0 507/510 (99%), AJ244265|AJ244265.1Trimmatostroma abietina 5.8S rRNA gene and . . . 965 0.0 503/507 (99%),AJ244231|AJ244231.1 Aureobasidium pullulans 5.8S rRNA gene and . . . 9650.0 503/507 (99%), AJ244235|AJ244235.1 Aureobasidium pullulans 5.8S rRNAgene and . . . 963 0.0 501/506 (99%) AJ244234|AJ244234.1 Aureobasidiumpullulans 5.8S rRNA gene and . . . 963 0.0 501/506 (99%)AF121285|AF121285.1 Aureobasidium pullulans strain ATCC48433 in . . .952 0.0 501/508 (98%) AF121281|AF121281.1 Aureobasidium pullulans strainATCC11942 in . . . 952 0.0 501/508 (98%) AJ244232|AJ244232.1Aureobasidium pullulans 5.8S rRNA gene and . . . 948 0.0 499/506 (98%)AF182377|AF182377.1 Hormonema sp. F-054, 764 internal transcribe . . .940 0.0 501/510 (98%) AF121284|AF121284.1 Aureobasidium pullulans strainATCC42457 in . . . 936 0.0 499/508 (98%) AJ244233|AJ244233.1Aureobasidium pullulans 5.8S rRNA gene and . . . 934 0.0 492/499 (98%)AJ244269|AJ244269.1 Aureobasidium pullulans 5.8S rRNA gene and . . . 9320.0 497/506 (98%) AJ244236|AJ244236.1 Aureobasidium pullulans 5.8S rRNAgene and . . . 932 0.0 497/506 (98%) AF121286|AF121286.1 Aureobasidiumpullulans var. melanigenum st . . . 920 0.0 497/508 (97%)AJ276061|AJ276061.1 Aureobasidium pullulans 5.8S rRNA gene and . . . 9140.0 497/505 (98%), AJ244252|AJ244252.1 Kabatiella lini 5.8S rRNA geneand internal . . . 906 0.0 496/508 (97%), AJ244251|AJ244251.1 Kabatiellacanlivora 5.8S rRNA gene and int . . . 825 0.0 487/508 (95%),AF013225|AF013225.1 Phaeocryptopus gaeumannii 18S ribosomal RNA . . .446 e−124 361/398 (90%), AJ244257|AJ244257.1 Pringsheimia smilacis 5.8SrRNA gene and in . . . 428 e−118 255/264 (96%), AJ244248|AJ244248.1Hormonema prunorum 5.8S rRNA gene and inter . . . 420 e−116 253/264(95%), AJ244245|AJ244245.1 Dothiora rhamni-alpinae 5.8S rRNA gene and .. . 420 e−116 252/264 (95%) AJ244242|AJ244242.1 Dothichiza pityophila5.8S rRNA gene and in . . . 418 e−115 246/255 (96%), AF182376|AF182376.1Kabatina juniperi internal transcribed spac . . . 416 e−115 251/262(95%), AF182375|AF182375.1 Hormonema sp. ATCC74360 internal transribe .. . 416 e−115 251/262 (95%), AJ244243|AJ244243.1 Dothiora cannabinae5.8S rRNA gene and inte . . . 412 e−113 252/264 (95%),AF027764|AF027764.1 Dothidea insculpta CBS 189.58 18S ribosomal . . .412 e−113 252/264 (95%), AF013226|AF013226.1 Kabatina thujae 18Sribosomal RNA gene, par . . . 412 e−113 251/264 (95%),AF027763|AF027763.1 Dothidea hippophaeos CBS 18658 18S ribosom . . . 404e−111 251/264 (95%), AJ278930|AJ278930.1 Hormonema dematioides 5.8S rRNAgene, 26S r . . . 402 e−110 244/255 (95%), AJ278929|AJ278929.1 Hormonemadematioides 5.8S rRNA gene, 26S r . . . 402 e−110 244/255 (95%),AJ278928|AJ278928.1 Hormonema dematioides 18S rRNA gene (partia . . .402 e−110 244/255 (95%), AJ278927|AJ278927.1 Hormonema dematioides 5.8SrRNA gene, 26S r . . . 402 e−110 244/255 (95%),

TABLE 10 Sequences producing significant alignments: (bits) ValueAJ278926|AJ278926.1 Hormonema dematioides 5.8S rRNA gene, 26S r . . .402  e−110 244/255 (95%), AJ278925|AJ278925.1 Hormonema dematioides 5.8SrRNA gene, 26S r . . . 402  e−110 244/255 (95%), AJ244262|AJ244262.1Sydowia polyspora 5.8S rRNA gene and intern . . . 402  e−110 244/255(95%), AJ244247|AJ244247.1 Hormonema macrosporum 5.8S rRNA gene and in .. . 402  e−110 244/255 (95%), AJ244244|AJ244244.1 Dothiora europaea 5.8SrRNA gene and intern . . . 402  e−110 252/263 (95%), AF182378|AF182378.1Hormonema sp. F-054,258 internal transcribe . . . 402  e−110 240/251(95%), AF013232|AF013232.1 Rhizosphaera kalkhoffii 18S ribosomal RNA g .. . 402  e−110 244/255 (95%), AF013228|AF013228.1 Hormonema dematioides18S ribosomal RNA gen . . . 402  e−110 244/255 (95%),AF260224|AF260224.1 Kabatina juniperi 18S ribosomal RNA, partia . . .396  e−109 250/264 (94%), AF013231|AF013231.1 Rhizosphaera kalkhoffii18S ribosomal RNA g . . . 389  e−106 244/256 (95%), AF121283|AF121283.1Aureobasidium pullulans strain ATCC16629 in . . . 379  e−103 238/251(94%), AF121282|AF121282.1 Aureobasidium pullulans strain ATCC16628 in .. . 379  e−103 238/251 (94%), AF013230|AF013230.1 Rhizosphaera pini 18Sribosomal RNA gene, p . . . 375  e−102 244/257 (94%),AF246930|AF246930.1 Botryosphaeria mamane isolate 97-59 18S rib . . .359 2e−97 214/225 (95%) AF246929|AF246929.1 Botryosphaeria mamaneisolate 97-58 18S rib . . . 359 2e−97 214/225 (95%) AF243410|AF243410.1Sphaeropsis sapinea isolate 215 18S ribosom . . . 345 2e−93 214/226(94%), AF243409|AF243409.1 Sphaeropsis sapinea isolate 411 18S ribosom .. . 345 2e−93 214/226 (94%), U28059|U28059.1 Sphaceloma fawcettii 18Sribosomal RNA and 26S . . . 339 1e−91 189/195 (96%) U28058|U28058.1Elsinoe fawcettii 18S ribosomal RNA and 26S rib . . . 339 1e−91 189/195(96%) AF297232|AF297232.1 Cercospora sorghi f. maydis Kenya 1 18S rib .. . 335 2e−90 187/193 (96%) AF297230|AF297230,1 Cercospora nicotianae18S ribosomal RNA gen . . . 335 2e−90 187/193 (96%) AF297229|AF297229.1Cercospora asparagi 18S ribosomal RNA gene . . . 335 2e−90 187/193 (96%)AF243394|AF243394.1 Botryosphaeria ribis isolate 968 18S ribos . . . 3352e−90 212/225 (94%), AF243393|AF243393.1 Botryosphaeria ribis isolate94-128 18S rib . . . 335 2e−90 212/225 (94%), AF079776|AF079776.1Phomopsis amaranthicola 18S ribosomal RNA g . . . 335 2e−90 187/193(96%) AB041245|AB041245.1 Guignardia laricina genes for 18S rRNA, ITS .. . 333 9e−90 189/196 (96%) AF297667|AF297667.1 Umbilicariamuehlenbergii isolate sm11004 1 . . . 331 4e−89 182/187 (97%)AF297666|AF297666.1 Umbilicaria muehlenbergii isolate sm11003 1 . . .331 4e−89 182/187 (97%) AF141190|AF141190.1 Neofabraea alba 18Sribosomal RNA gene, par . . . 331 4e−89 182/187 (97%)AF141189|AF141189.1 Neofabraea malicorticis 18S ribosomal RNA g . . .331 4e−89 182/187 (97%) AF141181|AF141181.1 Pezicola ocellata strainCBS267.39 18S ribo . . . 331 4e−89 182/187 (97%) AF096204|AF096204.1Umbilicaria muehlenbergii 18S ribosomal RNA . . . 331 4e−89 182/187(97%) AF083199|AF083199.1 Phialophora sp. p3901 18S ribosomal RNA, pa .. . 331 4e−89 182/187 (97%) AF383949|AF383949.1 Botryosphaeria quercuum18S ribosomal RNA g . . . 327 6e−88 190/197 (96%),

TABLE 11 Sequences producing significant alignments: (bits) ValueAJ276062|AJ276062.1 Aureobasidium pullulans 5.8S rRNA gene and . . .1043 0.0 526/526 (100%) AF182377|AF182377.1 Hormonema sp. F-054, 764internal transcribe . . . 1011 0.0 510/510 (100%) AF121284|AF121284.1Aureobasidium pullulans strain ATCC42457 in . . . 1007 0.0 508/508(100%) AF013229|AF013229.1 Aureobasidium pullulans 18S ribosomal RNA g .. . 1005 0.0 556/567 (98%), AJ244269|AJ244269.1 Aureobasidium pullulans5.8S rRNA gene and . . . 1003 0.0 506/506 (100%) AJ244236|AJ244236.1Aureobasidium pullulans 5.8S rRNA gene and . . . 1003 0.0 506/506 (100%)AF121286|AF121286.1 Aureobasidium pullulans var. melanigenum st . . .991 0.0 506/508 (99%) AF121285|AF121285.1 Aureobasidium pullulans strainATCC48433 in . . . 959 0.0 502/508 (98%) AF121281|AF121281.1Aureobasidium pullulans strain ATCC11942 in . . . 959 0.0 502/508 (98%)AJ244232|AJ244232.1 Aureobasidium pullulans 5.8S rRNA gene and . . . 9550.0 500/506 (98%) AJ276061|AJ276061.1 Aureobasidium pullulans 5.8S rRNAgene and . . . 954 0.0 502/505 (99%), AJ244265|AJ244265.1 Trimmatostromaabietina 5.8S rRNA gene and . . . 942 0.0 500/507 (98%),AJ244233|AJ244233.1 Aureobasidium pullulans 5.8S rRNA gene and . . . 9420.0 493/499 (98%) AJ244231|AJ244231.1 Aureobasidium pullulans 5.8S rRNAgene and . . . 942 0.0 500/507 (98%), AJ244235|AJ244235.1 Aureobasidiumpullulans 5.8S rRNA gene and . . . 932 0.0 497/506 (98%)AJ244234|AJ244234.1 Aureobasidium pullulans 5.8S rRNA gene and . . . 9320.0 497/506 (98%) AF121287|AF121287.1 Aureobasidium pullulans var.melanigenum st . . . 924 501/510 (98%),

TABLES 12 Sequences producing significant alignments: (bits) ValueAJ276062|AJ276062.1 Aureobasidium pullulans 5.8S rRNA gene and . . .1021 0.0 525/527 (99%), AF013229|AF013229.1 Aureobasidium pullulans 18Sribosomal RNA g . . . 997 0.0 554/567 (97%), AF182377|AF182377.1Hormonema sp. F-054,764 internal transcribe . . . 989 0.0 509/511 (99%),AF121284|AF121284.1 Aureobasidium pullulans strain ATCC42457 in . . .985 0.0 507/509 (99%), AJ244269|AJ244269.1 Aureobasidium pullulans 5.8SrRNA gene and . . . 981 0.0 505/507 (99%), AJ244236|AJ244236.1Aureobasidium pullulans 5.8S rRNA gene and . . . 981 0.0 505/507 (99%),AF121286|AF121286.1 Aureobasidium pullulans var. melanigenum st . . .969 0.0 505/509 (99%), AJ244265|AJ244265.1 Trimmatostroma abietina 5.8SrRNA gene and . . . 965 0.0 502/507 (99%) AJ244231|AJ244231.1Aureobasidium pullulans 5.8S rRNA gene and . . . 965 0.0 502/507 (99%)AJ276061|AJ276061.1 Aureobasidium pullulans 5.8S rRNA gene and . . . 9400.0 502/506 (99%), AF121287|AF121287.1 Aureobasidium pullulans var.melanigenum st . . . 940 0.0 502/510 (98%), AF121285|AF121285.1Aureobasidium pullulans strain ATCC48433 in . . . 938 0.0 501/509 (98%),AF121281|AF121281.1 Aureobasidium pullulans strain ATCC11942 in . . .938 0.0 501/509 (98%), AJ244232|AJ244232.1 Aureobasidium pullulans 5.8SrRNA gene and . . . 934 0.0 499/507 (98%), AY029406|AY029406.1 Astasialonga internal transcribed spacer 1 . . . 932 0.0 531/546 (97%),AJ244235|AJ244235.1 Aureobasidium pullulans 5.8S rRNA gene and . . . 9260.0 498/507 (98%), AJ244234|AJ244234.1 Aureobasidium pullulans 5.8S rRNAgene and . . . 926 0.0 498/507 (98%), AJ244233|AJ244233.1 Aureobasidiumpullulans 5.8S rRNA gene and . . . 920 0.0 492/500 (98%),AJ244252|AJ244252.1 Kabatiella lini 5.8S rRNA gene and internal . . .912 0.0 497/508 (97%), AJ244251|AJ244251.1 Kabatiella caulivora 5.8SrRNA gene and int . . . 872 0.0 492/508 (96%), AF013225|AF013225.1Phaeocryptopus gaeumannii 18S ribosomal RNA . . . 454 e−126 362/398(90%), AJ244257|AJ244257.1 Pringsheimia smilacis 5.8S rRNA gene and in .. . 436 e−121 256/264 (96%),

EXAMPLE 50

Into a 30 ml volume test tube was put 5.5 ml of YM medium (Difco) andsterilized at 121° C. for 20 minutes. After cooling, the medium wasinoculated with a platinum loopful of ADK-34 stored on a YPD-agar medium(Difco) slant and incubated at 26° C. for 4 days in a shake incubator toobtain a seed culture. Into another test tube was put 5.5 ml of Czapek'smedium (Difco; sucrose concentration: 3 wt %), sterilized, andinoculated with 500 μl of the ADK-34 seed culture (5% inoculation),followed by cultivation at 26° C. for 4 days in a shake incubator (300rpm). The culture was white and significantly viscous. After thecultivation, an equivalent amount of distilled water was added to theculture. The culture was autoclaved for 15 minutes and then centrifugedat 10,000 rpm for 15 minutes to obtain the culture solution (supernatantliquid) containing polysaccharides. The β-glucan content of the culturesolution and its purity with respect to polysaccharide pullulan weremeasured in accordance with the methods described in Analysis Examples 1and 3. As a result, the purity with respect to polysaccharide pullulanwas 100%, and the β-glucan yield based on the sugar, calculated from theβ-glucan content, was 44%. The culture solution had an absorbance of0.030 at 490 nm, which indicates suppression of coloration. The β-glucanwas found to have a molecular weight range of from 150,000 to 2,000,000as measured in accordance with the method of Analysis Example 2. Theculture solution was lyophilized to give sample L.

IFO-6353 was grown in the same manner as described above, and theβ-glucan content of the culture solution and the purity with respect topolysaccharide pullulan were measured in the same manner as describedabove. The purity with respect to polysaccharide pullulan was 40%, andthe yield of β-glucan based on the sugar was calculated to be 11%. Theabsorbance of the culture solution at 490 nm was 0.190, indicatingpigmentation.

EXAMPLE 51

In 100 ml of distilled water was dissolved 1.7 g of Yeast Nitrogen Basew/o Amino Acids and Ammonium Sulfate (Difco), and the solution wassterilized by filtration. Separately, 5 g of sodium nitrate wasdissolved in 500 ml of distilled water to prepare a sodium nitratesolution having a doubled concentration. Twelve weight percent carbonsource aqueous solutions were prepared using each of sucrose, glucose,fructose, soluble starch, and maltose (Wako Pure Chemical). The sodiumnitrate solution, aqueous carbon source solutions, and distilled waterwere each autoclaved at 121° C. for 20 minutes. Into a sterile test tubewere put 1 ml of the Yeast Nitrogen Base w/o Amino Acids and AmmoniumSulfate, 5 ml of the sodium nitrate solution, 2.5 ml of each of thecarbon source aqueous solutions, and 1.5 ml of distilled water to make10 ml to prepare media for examining carbon source utilization (checkingmedia).

Into a 30 ml volume test tube was put 5.5 ml of YM medium (Difco) andsterilized at 121° C. for 20 minutes. After cooling, the medium wasinoculated with a platinum loopful of ADK-34 stored on a YPD-agar medium(Difco) slant and incubated at 26° C. for 4 days in a shake incubator toobtain a seed culture. The ADK-34 seed culture was inoculated into eachof the above-prepared checking media, followed by cultivation at 26° C.for 5 days in a shake incubator (300 rpm). All the cultures were whiteand significantly viscous. After the cultivation, an equivalent amountof distilled water was added to each of the cultures. The culture wasautoclaved for 15 minutes and then centrifuged at 10,000 rpm for 15minutes to obtain the culture solution (supernatant liquid) containingpolysaccharides. The β-glucan content of the culture solution and itspurity with respect to polysaccharide pullulan were measured inaccordance with the methods described in Analysis Examples 1 and 3. Theresults of measurements are shown in Table 13 below. As is apparent fromTable 13, the purity with respect to polysaccharide pullulan was 90% orhigher, and the β-glucan yield based on the sugar was calculated torange from 20 to 46% with any of the carbon sources. The culturesolution had an absorbance of 0.026 to 0.041 at 490 nm, which indicatessuppression of coloration. The β-glucan was found to have a molecularweight range of from 150,000 to 2,000,000 as measured in accordance withthe method of Analysis Example 2. TABLE 13 Purity β-Glucan Yield basedAbsorbance of Culture Carbon Source (%) on Sugar (%) Solution (490 nm)Sucrose 100 46 0.031 Glucose 110 44 0.033 Fructose 104 20 0.031 Solublestarch 96 34 0.041 Maltose 94 29 0.026

EXAMPLE 52

ADK-34 was inoculated into YM medium and incubated at 26° C. for 3 daysto obtain 300 ml of a seed culture. Into a 5 liter volume jar fermentorequipped with impellers “FULLZONE” (B.E. Marubishi Co., Ltd.) were put 3liters of Czapek's medium and 300 g of sucrose. After sterilization andcooling, the medium was inoculated with 100 ml of the seed culture,followed by cultivation at 26° C. for 72 hours to obtain a culturemeasuring 3 liters. The culture (3 liters) was sterilized by heating at80° C. for 30 minutes, mixed with an equivalent amount of distilledwater, followed by mixing well. The mixture was centrifuged at 8000 rpm,for 15 minutes to get diluted culture. The resulting diluted culture wasfurther diluted 5-fold with distilled water. The absorbance of thediluted culture at 490 nm was 0.023. To 100 ml of the diluted culturewas added an equivalent amount of ethanol. The thus formed precipitatewas collected, washed with ethanol, and dissolved in 50 ml of distilledwater. The ethanol precipitation was repeated once more, and theresulting aqueous solution was put into a dialysis bag (molecular weightcut: 3000) and dialyzed against 10 times the volume of distilled waterto finally obtain 100 ml of a polysaccharide solution. The β-glucancontent was 25 mg/ml; the purity with respect to polysaccharide pullulanwas 95%; and the molecular weight of the β-glucan ranged 150,000 to2,000,000.

EXAMPLE 53 Beta-Glucan-Containing Fat and Oil Composition

A hundred parts of sample L and 100 parts of soybean oil were thoroughlymixed in a kneader. The mixture was allowed to stand at 60° C. for 10minutes and then cooled to room temperature to obtain a creamyβ-glucan-containing fat and oil composition of the invention. Thecomposition was found to have β-glucan uniformly dispersed therein.

INDUSTRIAL APPLICABILITY

The β-glucan-containing fat and oil composition of the present inventionhas β-glucan exhibiting excellent bioregulatory functions uniformlydispersed therein. Added to a food, etc., the composition provides thefood with uniformly dispersed β-glucan and also with enhanced taste,texture, stability, and the like. Use of the microorganisms according tothe present invention makes it possible to produce high-activity andhigh-quality β-glucan from inexpensive sugars such as sucrose with goodefficiency at high production rates.

1. A β-glucan-containing fat and oil composition characterized bycontaining a β-glucan of microorganism origin or basidiomycete origin.2. The β-glucan-containing fat and oil composition according to claim 1,wherein the β-glucan is one secreted out of fungi by cultivatingmicroorganisms or basidiomycetes.
 3. The β-glucan-containing fat and oilcomposition according to claim 1, wherein the β-glucan is culture cellsobtained by cultivating microorganisms or basidiomycetes.
 4. Theβ-glucan-containing fat and oil composition according to claim 1,wherein the microorganisms are yeast, lactic acid bacteria, Chlorella,algae or a microorganism belonging to the genus Aureobasidium.
 5. Theβ-glucan-containing fat and oil composition according to claim 1,wherein the microorganism is one of which the 18S rRNA gene contains thesequence of 1732 bases shown in Sequence Listing, SEQ ID No. 1 or a basesequence molecular-phylogenetically equivalent thereto based on the 18SrRNA gene base sequence and which is resistant to the antibioticcycloheximide and capable of secreting and producing β-glucan out offungi.
 6. The β-glucan-containing fat and oil composition according toclaim 1, wherein the microorganism is one of which the ITS-5.8S rRNAgene contains the sequence of 563 bases shown in Sequence Listing, SEQID No. 2 or a base sequence molecular-phylogenetically equivalentthereto based on the ITS-5.8S rRNA gene base sequence and which iscapable of secreting and producing β-glucan out of fungi.
 7. Theβ-glucan-containing fat and oil composition according to claim 1,wherein the β-glucan content is 0.01 to 500 parts by weight per 100parts by weight of the total of the components other than the β-glucan.8. A food containing the β-glucan-containing fat and oil compositionaccording to claim
 1. 9. A bakery product containing theβ-glucan-containing fat and oil composition according to claim
 1. 10. Aconfectionery product containing the β-glucan-containing fat and oilcomposition according to claim
 1. 11. A food having a prophylacticaction for habitual diseases containing the β-glucan-containing fat andoil composition according to claim
 1. 12. A drug having a prophylacticaction for habitual diseases containing the β-glucan-containing fat andoil composition according to claim
 1. 13. A processed rice, wheat, maizeor soybean product containing the β-glucan-containing fat and oilcomposition according to claim
 1. 14. A microorganism of which the 18SrRNA gene contains the sequence of 1732 bases shown in Sequence Listing,SEQ ID No. 1 or a base sequence molecular-phylogenetically equivalentthereto based on the 18S rRNA gene base sequence and which is resistantto the antibiotic cycloheximide and capable of secreting and producingβ-glucan out of fungi.
 15. A microorganism of which the ITS-5.8S rRNAgene contains the sequence of 563 bases shown in Sequence Listing, SEQID No. 2 or a base sequence molecular-phylogenetically equivalentthereto based on the ITS-5.8S rRNA gene base sequence and which iscapable of secreting and producing β-glucan out of fungi.
 16. Themicroorganism according to claim 15, which has resistance to theantibiotic cycloheximide.
 17. The microorganism according to claim 14,which is capable of secreting and producing β-glucan having at least aβ-1,3-D-glucopyranose bond in the structure out of fungi.
 18. Themicroorganism according to claim 14, which belongs to the genusAureobasidium.
 19. The microorganism according to claim 14, which is thestrain of Aureobasidium pullulans ADK-34 (FERM BP-8391).
 20. A processof producing β-glucan characterized by comprising culturing themicroorganism according to claim 14, secreting and producing β-glucanout of fungi.
 21. A process of producing β-glucan characterized bycomprising culturing a microorganism of which the ITS-5.8S rRNA geneexhibits sequence homology of at least 98% with the base sequence shownin Sequence Listing, SEQ ID No. 2, secreting and producing β-glucan outof fungi.
 22. The process of producing β-glucan according to claim 20,wherein culturing of the microorganism is carried out using a culturemedium containing saccharides as a carbon source.
 23. Beta-glucan havingat least a β-1,3-D-glucopyranose bond in the structure thereof which issecreted and produced out of fungi by culturing the strain ofAureobasidium pullulans ADK-34 (FERM BP-8391).